Treatment for vitiligo

ABSTRACT

Compounds I and II, as well as prodrugs, hydrates, solvates, N-oxides, salts and pharmaceutical compositions containing them, are useful for treating vitiligo. In certain embodiments, the compounds are provided in topical compositions.

CROSS REFERENCE TO RELATED APPLICATION

This claims the benefit of U.S. Provisional Application No. 61/700,153,filed Sep. 12, 2012, which is incorporated herein in its entirety byreference.

FIELD

The present disclosure relates to compounds, hydrates, solvates,prodrugs, salts, and N-oxides thereof, and pharmaceutical compositionscomprising the compounds, and methods of using these compounds andcompositions thereof in the treatment of vitiligo.

BACKGROUND

Vitiligo is a condition that causes depigmentation of skin, typically insections or patches, and affects about 1-2% of the world population.Vitiligo occurs when there is an absence of functional melanocytes(melanin-producing cells) in the skin. Vitiligo also can affect themucous membranes and the eye. There may be a genetic predisposition tovitiligo in some cases. The average age at vitiligo onset is about 20years, with onset most commonly observed between the ages of 10 and 30.

Vitiligo occurs most often on the face and extremities—typically thehands and wrists. Depigmentation also can occur around the mouth, eyes,nostrils, genitalia, and umbilicus. Depigmented patches are flat areasof normal-feeling skin, and may have a hyperpigmented edge. The edgestypically are well-defined but irregular. In trichrome vitiligo, thereis an intermediate zone of hypochromia between the achromic center andperipheral unaffected skin.

There are several clinical classifications of vitiligo. Segmentalvitiligo presents as one or more macules in a dermatomal orquasidermatomal pattern, and occurs most commonly in children. All othertypes of vitiligo are classified as non-segmental vitiligo, which ismost common. Focal vitiligo is characterized by depigmentation in onearea, or macule, such as the trigeminal nerve distribution. Other formsof non-segmental vitiligo often produce symmetric patches, sometimescovering large areas. Mucosal vitiligo affects only mucosal membranes.Generalized vitiligo may be acrofacial, in which depigmentation occurson the distal fingers and periorificial areas, or vulgaris, which ischaracterized by widely distributed, scattered patches. Universalvitiligo manifests as complete or nearly complete depigmentation, andfrequently is associated with multiple endocrinopathy syndrome. Theexact cause of melanocyte loss in vitiligo remains debatable, but recentobservations have pointed to a role for cellular immunity in thepathogenesis of vitiligo (see, for example, Wang et al. (2011) Th17Cells and Activated Dendritic Cells Are Increased in Vitiligo Lesions.PLoS ONE 6(4): e18907). Despite some advances in elucidating the originsof the disorder, current therapies, such as topical corticosteroids,topical immunomodulators and psoralen phototherapy have serious sideeffects and limited therapeutic utility. Vitiligo is a disfiguringdisease for which current therapies have proven unsatisfactory.

SUMMARY

Disclosed are compounds, prodrugs, corresponding salt forms, and methodsof using these compounds, and solvates, prodrugs and salt forms thereof,that can be used to provide therapeutic benefit to subjects who havevitiligo or prophylactically for subjects who are at risk of developingvitiligo. For the purposes of brevity in description, for any embodimentwhere a compound is referred to specifically, the embodiment alsoincludes hydrates, solvates, prodrugs, salt forms, and N-oxides of thecompound, and/or a pharmaceutical composition containing the compound.The compounds may be administered by any suitable method, such assystemically, topically, or ocularly. In particular examples thecompound is applied directly to depigmented areas of skin, for examplein a topical formulation.

One embodiment provides a compound I:

Another embodiment provides a particular prodrug of compound I, which iscompound II:

In one aspect, vitiligo is treated using an effective amount of compoundI, compound II, and combinations thereof, and/or an effective amount ofone or more pharmaceutical compositions that include the compound orcompounds. One embodiment provides a method of treating vitiligo byadministering to the subject an amount of compound I, compound II, or acombination thereof effective to treat vitiligo. In particular examples,the compound is administered in a topical formulation directly todepigmented skin, or localized areas including depigmented patches(e.g., the hands or face), without applying it to any substantial amountof unaffected skin.

In one aspect of the disclosed method, administration of one or more ofthe presently disclosed compounds is effective to cause at least partialregression, such as repigmentation of existing areas and/or reducedincidence of new areas, of the depigmented skin that characterize thedisease. In another aspect of the disclosed method, administration ofone or more of the disclosed compounds is effective to prevent vitiligoin a subject at risk of developing vitiligo.

In some examples, the subject is first determined to have vitiligo. Forexample, the subject displays one or more clinical and/orhistopathological features of vitiligo. In another example, the subjectis first determined to be at risk of developing vitiligo. For example,the subject may have a family history of vitiligo and be genetically atrisk of developing vitiligo, and/or the subject may have a history of adisease associated with vitiligo, such as thyroid disease.

In another aspect, compound I, compound II, a combination thereof, or acomposition comprising one or more of the compounds is administeredeither alone or in combination or adjunctively with one or moreadditional therapeutics, such as an anti-inflammatory, an antihistamine,an antibiotic, an antiviral, an emollient, an analgesic, systemicphototherapy, psoralen photochemotherapy, excimer laser therapy, thyroidhormone replacement medication, or any combination thereof.

In some examples, compound I, compound II, or a combination thereof isadministered alone or with another biologically active agent in atopical sunscreen agent (to minimize the exposure to ultraviolet lightthat may worsen vitiligo, cause sunburn, or cause skin cancer ofdepigmented areas). Yet other combination treatments can include the useof concomitant or adjunctive treatment including those treatmentsdescribed above.

Typically the disclosed compounds, when used for treating vitiligo, areadministered at least once daily, such as at least two, three or fourtimes daily, or are applied to the skin in a sustained release format(such as an adherent dispenser, for example a patch).

In another embodiment, a pharmaceutical formulation includes compound Iand/or compound II, either in parent or salt form, and at least onepharmaceutically acceptable excipient, diluent, preservative,stabilizer, or mixtures thereof.

These and other embodiments are described in more detail below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a prior art photograph that illustrates vitiligo on asubject's neck.

FIG. 2 is a prior art photograph that illustrates vitiligo on asubject's back.

FIG. 3 is a prior art photograph that illustrates vitiligo on asubject's hands.

DETAILED DESCRIPTION I. Abbreviations and Definitions

IL: Interleukin

JAK: Janus kinase

STAT: Signal transducer and activator of transcription

SYK: Spleen Tyrosine Kinase

As used herein, the following definitions shall apply unless otherwiseindicated.

“Corticosteroids” are steroid hormones that are produced in the adrenalcortex. Corticosteroids are involved in a wide range of physiologicsystems such as stress response, immune response and regulation ofinflammation, carbohydrate metabolism, protein catabolism, bloodelectrolyte levels, and behavior. Examples of corticosteroids includecortisol, prednisone and prednisolone. Corticosteroids can beadministered either orally, parenterally (for example by injection) orby direct topical application to a lesion on the skin, and they may becombined with the compounds of formula I and/or II in a combinationformulation. “Topical corticosteroids” are applied topically directly tothe skin, but long term use of topical corticosteroids causes unsightlyskin atrophy.

“Cutaneous” or “dermal” refers to the skin, which is the tissue formingthe outer covering of the vertebrate body. The skin (which is alsosometimes referred to as the “integumentary system”), in combinationwith the mucous membranes (particularly the oral, nasal, oral and eyelidmembranes) help protect the body from its external environment. The skinconsists of two layers (the dermis and epidermis), the outermost ofwhich may be covered in many animals (including humans) at least in partwith hair.

It is mainly protective and sensory in function, along with the mucousmembranes of the eye, nose and mouth.

“Epithelial surfaces” refers to tissue made up of epithelial cells thatcover the surfaces of the body. Epithelial surfaces include externalsurfaces such as the skin and mucosa of the mouth and nose, as well asthe linings of internal body surfaces.

“External” epithelial surfaces are those exposed to the surfaces of thebody (such as the skin, and the lining of the nose and mouth) and thatare accessible to direct application of creams or ointments to thesurface without the use of instrumentation (such as endoscopes orscalpels).

“Mucous membranes” (or “mucosa”) are linings of mostly endodermalorigin, covered in epithelium, which are involved in absorption andsecretion. They line cavities that are exposed to the externalenvironment and internal organs. They are continuous with skin atseveral locations, such as the nostrils, mouth, lips, eyelids, ears,genital area, and anus.

“Non-steroidal anti-inflammatory drug (NSAID)” is a type ofanti-inflammatory agent that works by inhibiting the production ofprostaglandins. NSAIDS exert anti-inflammatory, analgesic andantipyretic actions. Examples of NSAIDS include ibuprofen, ketoprofen,piroxicam, naproxen, sulindac, aspirin, choline subsalicylate,diflunisal, fenoprofen, indomethacin, meclofenamate, salsalate, tolmetinand magnesium salicylate. These agents can be administered eitherorally, parenterally (for example by injection) or by direct topicalapplication to an inflamed area, and they may be combined with thecompounds of formula I and/or II in a combination formulation.

“N-oxide” refers to a compound including a nitrogen bearing an oxyradical.

“Pharmaceutically acceptable salt” refers to a biologically compatiblesalt of a compound that can be used as a drug, which salts are derivedfrom a variety of organic and inorganic counter ions well known in theart.

“Pharmaceutically effective amount” or “therapeutically effectiveamount” refers to an amount of a compound sufficient to treat aspecified disorder or disease or one or more of its symptoms and/or toprevent the occurrence of the disease or disorder. “Treatment” includesarresting further advancement of a disease, as well as reversing thedisorder, inducing regression of lesions, or in some examples curing thedisorder.

“Prodrug” refers to compounds that are transformed in vivo to yield theparent compound, for example, by hydrolysis in the gut or enzymaticconversion in blood. Common examples include, but are not limited to,ester and amide forms of a compound having an active form bearing acarboxylic acid moiety. Examples of pharmaceutically acceptable estersof the presently disclosed compounds include, but are not limited to,alkyl esters (for example with between about one and about six carbons)where the alkyl group is a straight or branched chain. Acceptable estersalso include cycloalkyl esters and arylalkyl esters such as, but notlimited to benzyl. Examples of pharmaceutically acceptable amides of thedisclosed compounds include, but are not limited to, primary amides, andsecondary and tertiary alkyl amides (for example with between about oneand about six carbons). Amides and esters of the disclosed compounds canbe prepared according to conventional methods. A thorough discussion ofprodrugs is provided in T. Higuchi and V. Stella, “Pro-drugs as NovelDelivery Systems,” Vol 14 of the A.C.S. Symposium Series, and inBioreversible Carriers in Drug Design, ed. Edward B. Roche, AmericanPharmaceutical Association and Pergamon Press, 1987, both of which areincorporated herein by reference for all purposes.

“Solvate” refers to a complex formed by combination of solvent moleculeswith molecules or ions of the solute. The solvent can be an organiccompound, an inorganic compound, or a mixture of both. Some examples ofsolvents include, but are not limited to, methanol,N,N-dimethylformamide, tetrahydrofuran, dimethylsulfoxide, and water.The compounds described herein can exist in unsolvated as well assolvated forms with solvents, pharmaceutically acceptable or not, suchas water, ethanol, and the like. Solvated forms of the presentlydisclosed compounds are contemplated herein and are encompassed by theinvention, at least in generic terms.

“Subject” refers to humans and non-human subjects.

“Topical” delivery refers to application of a drug-containingformulation to the skin to directly treat cutaneous disorders or thecutaneous manifestations of a disease with the intent of substantiallydirecting the pharmacological effect of the drug to the surface of theskin or within the skin. Topical dosage forms are typically semi-solidsystems, but can include a variety of other dosage forms such as foams,sprays, medicated powders, solutions and medicated adhesive systems.Topical delivery includes external topical agents that are spread,sprayed, or otherwise dispersed on cutaneous tissues to cover theaffected area, or internal topical agents that are applied to the mucousmembranes orally, vaginally, or on anorectal tissues for local activity.The topical drugs disclosed herein can be administered in any topicaldosage form, for example as a solid (powder, aerosol or plaster); liquid(lotion, liniment, solution, emulsion, suspension, aerosol) orsemi-solid (ointment, cream, paste, gel, jelly or suppository).

As used herein, and as well understood in the art, “treatment” is anapproach for obtaining beneficial or desired results, including clinicalresults. For the purposes of this invention, beneficial or desiredresults can include one or more, but are not limited to, alleviation oramelioration of one or more symptoms, diminishment of extent of acondition, including a disease, stabilized (i.e., not worsening) stateof a condition, including diseases, preventing spread of disease, delayor slowing of condition, including disease, progression, amelioration orpalliation of the condition, including disease, state, and remission(whether partial or total), whether detectable or undetectable.

“Vitiligo” is a condition characterized by loss of cutaneous melanocytesand/or abnormal melanocyte function.

II. Compounds

Disclosed are compounds, solvates, prodrugs, corresponding salt forms,and methods of using these compounds, hydrates, solvates, prodrugs andsalt forms in the treatment and/or prevention of vitiligo. For thepurposes of brevity in description, for any embodiment where compound Iand compound II are referred to specifically, the embodiment alsoincludes hydrates, solvates, prodrugs, salt forms, and N-oxides of thecompound, and/or a pharmaceutical composition containing compound Iand/or compound II.

Compounds I and II and pharmaceutical compositions containing them aredescribed in more detail below. Compound I is also referred to asN2-(3-aminosulfonyl-4-methylphenyl)-5-fluoro-N4-[4-(prop-2-ynyloxy)phenyl]-2,4-pyrimidinediamine.Compound II is also referred to as5-fluoro-N2-(4-methyl-3-propionylaminosulfonylphenyl)-N4-[4-(prop-2-ynyloxy)phenyl]-2,4-pyrimidinediamine.

One of ordinary skill in the art will appreciate that compound II is aprodrug of compound I, and that compound II need not necessarily bepharmacologically inactive until converted into compound I, as currentlyunderstood. The mechanism by which the propionyl progroup metabolizes isnot critical, and can be caused by, for example, hydrolysis under theacidic conditions of the stomach, and/or by enzymes present in thetissues and/or organs of the body, such as the skin, mucous membranes,saliva, tears, and including such enzymes as, for example, esterases,amidases, lipolases, phosphatases including ATPases and kinases,cytochrome P450's of the liver, and the like. In particular embodimentsdescribed herein, compounds I and/or II are used to treat vitiligo, andmay therefore be administered directly to the skin. If an initiallyinactive prodrug is administered, it can be activated subsequent toadministration, such as by enzymes (such as esterases) in the skin, ortopically administered with another agent that activates the drug (forexample, a reservoir of an activating substance in a patch, or anadditional agent that is mixed with the prodrug prior to topicalapplications). In some embodiments, administration may include not onlytopical administration but also injection and the like, for exampleintradermal injection. Alternatively, these active agents may beadministered systemically.

One of ordinary skill in the art will appreciate that compounds I and IImay exist as tautomers, conformational isomers and/or geometric isomers.It should be understood that the disclosure encompasses any tautomeric,conformational isomeric and/or geometric isomeric forms of the compoundsas well as mixtures of these various different isomeric forms.

Atropisomers are stereoisomers resulting from hindered rotation aboutsingle bonds where the barrier to rotation is high enough to allow forthe isolation of the conformers (Eliel, E. L.; Wilen, S. H.Stereochemistry of Organic Compounds; Wiley & Sons: New York, 1994;Chapter 14). Atropisomerism is significant because it introduces anelement of chirality in the absence of stereogenic atoms. The inventionis meant to encompass atropisomers, for example in cases of limitedrotation about bonds between the 2,4-pyrimidinediamine core structureand groups attached thereto or for example about bonds between thesulfonamide and the phenyl ring to which it is attached.

Compounds I and II may be in the form of salts. Such salts include saltssuitable for pharmaceutical uses (“pharmaceutically-acceptable salts”),salts suitable for veterinary uses, etc. Such salts may be derived fromacids or bases, as is well-known in the art. Exemplary salts describedherein are sodium salts, potassium salts, arginine salts, choline saltsand calcium salts, but generically any pharmaceutically acceptable saltmay be used for methods described herein. Because compound I andcompound II have both basic groups, for example pyrimidine nitrogens,and acidic groups, for example acidic protons on the sulfonamide and/orthe nitrogens at N2 and N4 of the pyrimidinediamine system, thesecompounds can form pharmaceutically acceptable acid or base additionsalts.

Generally, pharmaceutically acceptable salts are those salts that retainsubstantially one or more of the desired pharmacological activities ofthe parent compound and which are suitable for administration to humans.Pharmaceutically acceptable salts include acid addition salts formedwith inorganic acids or organic acids. Inorganic acids suitable forforming pharmaceutically acceptable acid addition salts include, by wayof example and not limitation, hydrohalide acids (for example,hydrochloric acid, hydrobromic acid, hydroiodic acid, etc.), sulfuricacid, nitric acid, phosphoric acid, and the like. Organic acids suitablefor forming pharmaceutically acceptable acid addition salts include, byway of example and not limitation, acetic acid, trifluoroacetic acid,propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolicacid, oxalic acid, pyruvic acid, lactic acid, malonic acid, succinicacid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid,palmitic acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamicacid, mandelic acid, alkylsulfonic acids (for example, methanesulfonicacid, ethanesulfonic acid, 1,2-ethane-disulfonic acid,2-hydroxyethanesulfonic acid, etc.), arylsulfonic acids (for example,benzenesulfonic acid, 4-chlorobenzenesulfonic acid,2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonicacid, etc.), 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid,glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid,tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamicacid, hydroxynaphthoic acid (xinafoic acid), salicylic acid, stearicacid, muconic acid, and the like.

Pharmaceutically acceptable salts also include salts formed when anacidic proton present in the parent compound is either replaced by ametal ion (for example, an alkali metal ion (e.g., sodium or potassium),an alkaline earth metal ion (e.g., calcium or magnesium), or an aluminumion) or coordinates with an organic base (for example, ethanolamine,diethanolamine, triethanolamine, N-methylglucamine, morpholine,piperidine, dimethylamine, diethylamine, triethylamine, ammonia, etc.).

Salts of amine groups may comprise quaternary ammonium salts in whichthe amino nitrogen atom carries a suitable organic group such as analkyl, alkenyl, alkynyl or aralkyl moiety.

The pharmaceutically acceptable salts described herein may be formed byconventional means, such as by reacting the free base form of theproduct with one or more equivalents of the appropriate acid in asolvent or medium in which the salt is insoluble, or in a solvent suchas water which is removed in vacuo or by freeze drying or by exchangingthe anions of an existing salt for another anion on a suitable ionexchange resin.

Compounds I and II, as well as the salts thereof, may also be in theform of solvates, for example hydrates, and N-oxides, as are well-knownin the art.

III. Pharmaceutical Compositions

Pharmaceutical compositions comprising compounds I and II describedherein can be manufactured by means of conventional mixing, dissolving,granulating, dragee-making, levigating, emulsifying, encapsulating,entrapping or lyophilization processes. The compositions can beformulated in a conventional manner using one or more physiologicallyacceptable carriers, diluents, excipients or auxiliaries whichfacilitate processing of the active compounds into preparations whichcan be used pharmaceutically, and particularly locally or topically.

Compounds I and II can be formulated in the pharmaceutical compositionsper se, or in the form of a prodrug, hydrate, solvate, N-oxide orpharmaceutically acceptable salt, as described herein. Typically, suchsalts are more soluble in aqueous solutions than the corresponding freeacids and bases, but salts having lower solubility than thecorresponding free acids and bases may also be formed.

In one embodiment, a pharmaceutical formulation comprises compound Iand/or compound II, and at least one pharmaceutically acceptableexcipient, diluent, preservative, or stabilizer, or mixtures thereof. Inone embodiment, the compounds are provided as non-toxic pharmaceuticallyacceptable salts as noted previously.

Compounds I and II may be administered by any suitable method, such asoral, parenteral (for example, intramuscular, intraperitoneal,intravenous, ICV, intracisternal injection or infusion, subcutaneousinjection, or implant), inhalation spray, nasal, vaginal, rectal,sublingual, urethral (for example, urethral suppository) or topicalroutes of administration (for example, gel, ointment, cream, aerosol,etc.). Compounds I and II may be formulated, alone or together, insuitable dosage unit formulations containing conventional non-toxicpharmaceutically acceptable carriers, adjuvants, excipients and vehiclesappropriate for each route of administration. In addition to thetreatment of warm-blooded animals such as mice, rats, horses, cattle,sheep, dogs, cats, monkeys, etc., the compounds described herein can beused for treating humans.

The pharmaceutical compositions for the administration of compounds Iand II may conveniently be presented in dosage unit form and may beprepared by any of the methods well known in the art of pharmacy. Thepharmaceutical compositions can be, for example, prepared by uniformlyand intimately bringing the active ingredient into association with aliquid carrier or a finely divided solid carrier or both, and then, ifnecessary, shaping the product into the desired formulation and/orplacing it in appropriate packaging. In topical formulations of thedisclosed compounds, the formulation is placed in an appropriatecontainer (such as a squeeze-tube with a cap for dispensing ointmentsand creams). Alternatively, the dispenser may include a device fordispensing unit dosages of the drug (such as a bottle or dropper thatdispenses a controlled pre-determined dosage of the drug to a targetarea). In the pharmaceutical composition the active object compound isincluded in an amount sufficient to produce the desired therapeuticeffect. For example, pharmaceutical compositions described herein maytake a form suitable for virtually any mode of administration,including, for example, topical, ocular, oral, buccal, systemic, nasal,injection, transdermal, rectal, vaginal, etc., or a form suitable foradministration by inhalation or insufflation.

For topical administration, the compound(s) or prodrug(s) may beformulated as solutions, gels, ointments, creams, suspensions, etc. asare well-known in the art. In addition to being suitable foradministration to the skin, the solutions, gels, ointments, creams andsuspensions are also well-suited for administration directly to the eye.One embodiment is a pharmaceutical formulation comprising compound Iand/or compound II, where the formulation is selected from a solution, agel, an ointment, a cream and a suspension. In one aspect, suchformulations formulated for topical administration include atherapeutically effective amount of a compound I and/or compound II or apharmaceutically acceptable salt thereof, such as a hydrochloride saltor a besylate salt in the case of compound I and, by way of example, alysine, choline or arginine salt of compound II. Particular embodimentsof formulations for use in the methods described herein include atherapeutically effective amount of the compound, a topical base, anantioxidant, an emollient, and an emulsifier. A person of skill in theart will appreciate that a therapeutically effective amount of thecompound may vary, but typically a concentration ranging from 0.1% to10% (w/w) will provide a therapeutically effective amount of thecompound to the subject.

The topical base may comprise polyethylene glycol having a selectedmolecular weight. Particular embodiments comprise a polyethylene glycolhaving a molecular weight of from 3000 to 8000 Daltons as a topicalbase.

In certain embodiments, the formulation is an ointment, and may furtherinclude a water-miscible solvent, such as a polyalkylene glycol havingan average molecular weight of from 200 Daltons to 600 Daltons. Incertain embodiments the water-miscible solvent comprises PEG-400, andeven more particularly PEG-400 substantially free of impurities. Incertain embodiments, PEG-400 substantially free of impurities comprisesless than 65 ppm formaldehyde, less than 10 ppm formaldehyde, or 1 ppmor less formaldehyde.

Topical formulations for use as described herein also can include apenetration enhancer, such as dimethyl isosorbide, propylene glycol, orcombinations thereof; an emollient, such as water; a surfactant, such assorbitan monostearate, a polyethylene glycol monostearate,D-α-tocopheryl polyethylene glycol 1000 succinate, a compositioncomprising glycol stearate/PEG32 stearate/PEG6 stearate, andcombinations of surfactants; an antioxidant, such as butylatedhydroxyanisole, butylated hydroxytoluene, ascorbic acid, α-tocopherol,and combinations thereof, with particular embodiments comprisingbutylated hydroxytoluene as an antioxidant; and an optional colorant,such as 0.05% to 0.25% (w/w) caramel colorant.

For particular embodiments, a concentration ranging from 0.1% to 10%(w/w) provides a therapeutically effective amount of the compound to thesubject, and the pharmaceutical formulation further comprises: from 15%to 40% (w/w) of a topical base, such as a polyalkylene glycol with anaverage molecular weight of from 4000 to 5000 Daltons; from 25% to 50%(w/w) of a water-miscible solvent, such as a polyalkylene glycol with anaverage molecular weight of from 300 to 500 Daltons; from 10% to 20%(w/w) of a penetration enhancer, such as dimethyl isosorbide; from 3% to15% (w/w) of an emollient, such as water; from 3% to 9% (w/w) of asurfactant, such as polyethylene glycol monostearate; and from 0.5% to1.5% (w/w) butylated hydroxytoluene as an antioxidant.

Another embodiment of the pharmaceutical formulation comprises from 0.2%to 6% (w/w) of compound I or a pharmaceutically acceptable salt thereof;30% to 40% (w/w) polyethylene glycol with an average molecular weight offrom 4000 to 5000 Daltons; from 30% to 40% (w/w) polyethylene glycolwith an average molecular weight of from 300 to 500 Daltons; 15% (w/w)dimethyl isosorbide; 3 to 5% (w/w) water; 5% (w/w) polyethylene glycolmonostearate; 1% (w/w) butylated hydroxytoluene; and 0.05% caramel.

Yet another embodiment of the pharmaceutical formulation comprises 1%(w/w) compound I; 25% to 40% (w/w) polyethylene glycol with an averagemolecular weight of 4500 Daltons; and 30% to 45% (w/w) polyethyleneglycol with an average molecular weight of 400 Daltons.

Yet another embodiment of the pharmaceutical formulation comprises 3%(w/w) compound I; 32% (w/w) polyethylene glycol with an averagemolecular weight of 4500 Daltons; and 38% to 42% (w/w) polyethyleneglycol with an average molecular weight of 400 Daltons.

Yet another embodiment of the pharmaceutical formulation comprises 6%(w/w) compound I; 35% (w/w) polyethylene glycol with an averagemolecular weight of 4500 Daltons; and 33% to 35% (w/w) polyethyleneglycol with an average molecular weight of 400 Daltons.

In one embodiment, the formulation is a solution. In another embodiment,the formulation is a gel. In another embodiment, the formulation is asuspension. In yet another embodiment, the formulation is a cream orointment. One embodiment is any of the aforementioned formulations in akit for topical or local administration. In one embodiment, theformulation is a liquid, for example a homogeneous liquid or asuspension, sold in a bottle which dispenses the formulation as drops ora liquid film (for example from an applicator tip that contacts a targetarea of the skin to dispense the liquid substantially only on a targetarea of the skin to be treated). In one embodiment, the formulation is acream or ointment, sold in a tube which dispenses the formulation to atarget area of the skin. In another embodiment, the compound is providedin a viscous liquid (such as carboxylmethylcellulose,hydroxypropylmethycellulose, polyethylene glycol, glycerin, polyvinylalcohol, or oil containing drops) for rubbing into the skin orinstilling in the eye. The formulations may have preservatives or bepreservative-free (for example in a single-use container).

Compounds I and II can be used for manufacturing a composition ormedicament, including medicaments suitable for topical administration,such as creams, ointments, jellies, gels, solutions or suspensions, etc.In certain embodiments, compounds I and II may be formulated for topicaladministration with polyethylene glycol (PEG). These formulations mayoptionally comprise additional pharmaceutically acceptable ingredientssuch as diluents, stabilizers and/or adjuvants. In particularembodiments, the topical formulations are formulated for the treatmentof vitiligo.

Systemic formulations include those designed for administration byinjection, for example, subcutaneous, intravenous, intramuscular,intrathecal or intraperitoneal injection, as well as those designed fortransdermal, transmucosal oral or pulmonary administration.

Useful injectable preparations include sterile suspensions, solutions oremulsions of the active compound(s) in aqueous or oily vehicles. Thecompositions may also contain formulating agents, such as suspending,stabilizing and/or dispersing agent or activating agents for activatingthe prodrug. The formulations for injection may be presented in unitdosage form, for example, in ampules or in multidose containers, and maycontain added preservatives. They may also be provided in syringes, forexample syringes with needles from injection of the drug into the skin,for example directly into a depigmented area of skin.

Alternatively, the injectable formulation may be provided in powder formfor reconstitution with a suitable vehicle, including but not limited tosterile pyrogen free water, buffer, dextrose solution, etc., before use.The powder can include an activating agent for a prodrug, whichactivates the prodrug when the powder is solubilized in a vehicle. Tothis end, the active compound(s) may be dried by any art-knowntechnique, such as lyophilization, and reconstituted prior to use.

For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants are knownin the art, and include dimethyl sulfoxide (DMSO) and dimethylisosorbide. However, the penetrants can also be used to improve deliveryof the active agents into the skin.

For oral administration, the pharmaceutical compositions may take theform of, for example, lozenges, tablets or capsules prepared byconventional means with pharmaceutically acceptable excipients such asbinding agents (for example, pregelatinised maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (forexample, lactose, microcrystalline cellulose or calcium hydrogenphosphate); lubricants (for example, magnesium stearate, talc orsilica); disintegrants (for example, potato starch or sodium starchglycolate); or wetting agents (for example, sodium lauryl sulfate). Thetablets may be coated by methods well known in the art with, forexample, sugars, films or enteric coatings. Additionally, thepharmaceutical compositions containing the 2,4-substitutedpyrimidinediamine as active ingredient or prodrug thereof in a formsuitable for oral use, may also include, for example, troches, lozenges,aqueous or oily suspensions, dispersible powders or granules, emulsions,hard or soft capsules, or syrups or elixirs. Compositions intended fororal use may be prepared according to any method known to the art forthe manufacture of pharmaceutical compositions and such compositions maycontain one or more agents selected from the group consisting ofsweetening agents, flavoring agents, coloring agents and preservingagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets contain the active ingredient (including prodrug)in admixture with non-toxic pharmaceutically acceptable excipients whichare suitable for the manufacture of tablets. These excipients may be forexample, inert diluents, such as calcium carbonate, sodium carbonate,lactose, calcium phosphate or sodium phosphate; granulating anddisintegrating agents (for example, corn starch, or alginic acid);binding agents (for example starch, gelatin or acacia); and lubricatingagents (for example magnesium stearate, stearic acid or talc). Thetablets may be uncoated or they may be coated by known techniques todelay disintegration and absorption in the gastrointestinal tract andthereby provide a sustained action over a longer period. For example, atime delay material such as glyceryl monostearate or glyceryl distearatemay be employed. They may also be coated by the techniques described inthe U.S. Pat. Nos. 4,256,108; 4,166,452; and 4,265,874 to form osmotictherapeutic tablets for control release. The pharmaceutical compositionsdescribed herein may also be in the form of oil-in-water emulsions.

Liquid preparations for oral administration may take the form of, forexample, elixirs, solutions, syrups or suspensions, or they may bepresented as a dry product for constitution with water or other suitablevehicle before use. Such liquid preparations may be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (for example, sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (for example, lecithin oracacia); non-aqueous vehicles (for example, almond oil, oily esters,ethyl alcohol, Cremophore® or fractionated vegetable oils); andpreservatives (for example, methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts,preservatives, flavoring, coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound or prodrug, as is well known.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For rectal and vaginal routes of administration, the active compound(s)may be formulated as solutions (for retention enemas) suppositories orointments containing conventional suppository bases such as cocoa butteror other glycerides.

The active compound(s) or prodrug(s) can be conveniently delivered inthe form of an aerosol spray from pressurized packs or a nebulizer withthe use of a suitable propellant, for example, dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, fluorocarbons, carbondioxide or other suitable gas. When administering a pro-drug, it can beco-delivered and mixed thereby with an activating agent, for example toactive compound II to compound I. In the case of a pressurized aerosol,the dosage unit may be determined by providing a valve to deliver ametered amount. Capsules and cartridges for use in an inhaler orinsufflator (for example capsules and cartridges comprised of gelatin)may be formulated containing a powder mix of the compound and a suitablepowder base such as lactose or starch.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleaginous suspension. This suspension may beformulated according to the known art using those suitable dispersing orwetting agents and suspending agents which have been mentioned above.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally-acceptable diluent orsolvent. Among the acceptable vehicles and solvents that may be employedare water, Ringer's solution and isotonic sodium chloride solution.Compounds I and II may also be administered in the form of suppositoriesfor rectal or urethral administration of the drug. In particularembodiments, the compounds may be formulated as urethral suppositories,for example, for use in the treatment of fertility conditions,particularly in males, for example, for the treatment of testiculardysfunction.

According to the invention, 2,4-substituted pyrimidinediamine compoundscan be used for manufacturing a composition or medicament, includingmedicaments suitable for rectal or urethral administration. Theinvention also relates to methods for manufacturing compositionsincluding 2,4-substituted pyrimidinediamine compounds in a form that issuitable for urethral or rectal administration, including suppositories.

Included among the devices which may be used to administer particularexamples of compounds I and II are those well-known in the art, such as,metered dose inhalers, liquid nebulizers, dry powder inhalers, sprayers,foamers, thermal vaporizers, and the like. Other suitable technology foradministration of particular 2,4-substituted pyrimidinediamine compoundsincludes electrohydrodynamic aerosolizers. Sprays, aerosols,sponge-tipped applicators and foam dispensers can be used to administerthe compounds, either per se or in formulations, directly to the skin,or by intradermal injection directly into depigmented patches caused byvitiligo.

Typically formulations for skin administration contain apharmaceutically effective amount of a 2,4-pyrimidinediamine compounddisclosed herein, such as from about 0.0001% to about 10% or more byweight (w/w). In certain formulations, the pharmaceutically effectiveamount of the compound is 0.0003% to about 0.1% (w/w), such as fromabout 0.003% to about 0.5% (w/w), from about 0.01% to about 0.03% (w/w),or from about 0.1% to about 10% (w/w). In other examples, the compoundis present in at least 2%, 3% or 5% (w/w).

In certain examples an ophthalmic composition containing a presentlydisclosed 2,4-pyrimidinediamine compound for ocular administration(e.g., for vitiligo of the eye) includes a tonicity agent, a buffer, orboth. In certain examples of ophthalmic compositions the tonicity agentis a simple carbohydrate or a sugar alcohol. As is known to one ofordinary skill in the art, tonicity agents may be used in the presentcompositions to adjust the tonicity of the composition, preferably tothat of normal tears. Examples of suitable tonicity agents include,without limitation sodium chloride, potassium chloride, magnesiumchloride, calcium chloride, carbohydrates, such as dextrose, fructose,galactose, polyols, such as sugar alcohols, including by way of example,mannitol, sorbitol, xylitol, lactitol, isomalt, maltitol andcombinations thereof. Compositions containing a buffer contain, in someexamples, a phosphate, citrate, or both.

The 2,4-substituted pyrimidinediamine compound(s) or prodrug(s)described herein, or compositions thereof, will generally be used in anamount effective to treat or prevent vitiligo. The compound(s) may beadministered therapeutically to achieve therapeutic benefit orprophylactically to achieve prophylactic benefit. By therapeutic benefitis meant eradication or amelioration of vitiligo and/or eradication oramelioration of one or more of the symptoms associated with vitiligosuch that the patient reports an improvement in feeling or condition,notwithstanding that the patient may still be afflicted with vitiligo.For example, administration of a compound to a patient suffering fromvitiligo provides therapeutic benefit not only when the underlyingdepigmented patch is eradicated or ameliorated, but also when halting orslowing the progression of the disease, regardless of whetherimprovement in symptoms is realized.

For prophylactic administration, the compound may be administered to apatient at risk of developing vitiligo, such as those with a familyhistory of vitiligo or premature graying of the hair. Other risk factorscan be identified by those of skill in the art and may include Addison'sdisease, hypothyroidism, hyperthyroidism, alopecia greata, perniciousanemia, psoriasis, or adult-onset diabetes. Alternatively, prophylacticadministration may be applied to avoid the progression of symptoms in apatient diagnosed with vitiligo.

The amount of compound administered will depend upon a variety offactors, including, for example, the particular type of vitiligo beingtreated, the mode of administration, the severity of the vitiligo, theage and weight of the patient, the bioavailability of the particularactive compound, etc. Determination of an effective dosage is wellwithin the capabilities of those skilled in the art. A skilledpractitioner will be able to determine the optimal dose for a particularindividual. Effective dosages may be estimated initially from in vitroassays. For example, an initial dosage for use in animals may beformulated to achieve a circulating blood or serum concentration ofactive compound that is at or above an IC₅₀ of the particular compoundas measured in an in vitro assay, such as the in vitro assays describedin Examples 2 and 3 herein. Similarly, an initial dosage of prodrug forsystemic use in animals may be formulated to achieve a circulating bloodor serum concentration of the metabolite active compound that is at orabove an IC₅₀ of the particular compound in an in vitro assay.Calculating dosages to achieve such circulating blood or serumconcentrations taking into account the bioavailability of the particularcompound is well within the capabilities of skilled artisans. Forguidance, the reader is referred to Fingl & Woodbury, “GeneralPrinciples,” In: Goodman and Gilman's The Pharmaceutical Basis ofTherapeutics, Chapter 1, pp. 1-46, latest edition, Pergamon Press, andthe references cited therein.

Initial dosages can also be estimated from in vivo data, using animalmodels such as those disclosed in Example 7. Animal models useful fortesting the efficacy of compounds to treat or prevent the variousdiseases described above are well-known in the art. Dosage amounts forsystemic administration will typically be in the range of from about0.0001 or 0.001 or 0.01 mg/kg/day to about 100 mg/kg/day, but may behigher or lower, depending upon, among other factors, the activity ofthe compound, its bioavailability, the mode of administration andvarious factors discussed above. Systemic dosage amount and interval maybe adjusted individually to provide plasma levels of the compound(s)which are sufficient to maintain therapeutic or prophylactic effect. Forexample, the compounds may be administered once per week, several timesper week (for example, every other day), once per day or multiple timesper day, depending upon, among other things, the mode of administration,the specific indication being treated and the judgment of theprescribing physician. In cases of local administration or selectiveuptake, such as local topical administration, the effective localconcentration of active compound(s) may not be related to plasmaconcentration. Skilled artisans will be able to optimize effective localdosages without undue experimentation. In view of the much highertherapeutic index of topical administration to the skin, dosages can beincreased beyond general systemic dosages without significant additionalconcern for side-effects and toxicities.

The foregoing disclosure pertaining to the dosage requirements for the2,4-substituted pyrimidinediamine compounds is pertinent to dosagesrequired for prodrugs, with the realization, apparent to the skilledartisan, that the amount of prodrug(s) administered will also dependupon a variety of factors, including, for example, the bioavailabilityof the particular prodrug(s), the conversion rate and efficiency intoactive drug compound under the selected route of administration,co-administration of an activating agent, etc. Determination of aneffective dosage of prodrug(s) for a particular use and mode ofadministration is well within the capabilities of those skilled in theart.

For topical or ocular administration, effective dosages may be thosewhere no significant systemic circulation of the compounds results fromadministration to the skin or eye, for example, where a topicalformulation is applied directly to a depigmented patch and a verylocalized dose is utilized prior to significant systemic circulation.

IV. Methods

Disclosed herein are 2,4-substituted pyrimidinediamine compounds I andII, prodrugs, solvates, salts, N-oxides and pharmaceutical compositionsthereof, for use in treating and/or preventing vitiligo. In particular,compounds I and/or II, are administered alone or in combination withother agents. In some examples, compositions comprising compound II mayinclude activating agents to activate the prodrug compound II tocompound I.

Compounds I and II (at least as a source of compound I) are potent, andthus can be administered locally (for example topically or by injectionto the skin or mucous membrane) at very low doses, thus minimizingsystemic adverse effects.

Compounds I and II are potent and selective inhibitors of JAK kinasesand in particular JAK1/3-dependent cytokine signaling operative in T-and B-cells and Syk-dependent signaling in macrophages, dendritic cells,and B-cells. For example, Compound I has a half maximal effectiveconcentration (EC50) in human cell-based assays against JAK3 and Syk inthe range of 0.18 μM and 0.14 μM (Deuse et al. Transplantation 85(6)885-892), respectively, and has little or no activity on other cytokine(IL-1β and TNFα) or receptor tyrosine kinase (RTK) signaling, and is nota broad inhibitor of cell proliferation.

Compound I is particularly selective for cytokine signaling pathwayscontaining JAK3. As a consequence of this activity, the compounds may beused in a variety of in vitro, in vivo and ex vivo contexts to regulateor inhibit JAK kinase activity, signaling cascades in which JAK kinasesplay a role, and the biological responses effected by such signalingcascades. For example, in one embodiment, the compounds may be used toinhibit JAK kinase, either in vitro or in vivo, in virtually any celltype expressing the JAK kinase (such as hematopoietic cells). They mayalso be used to regulate signal transduction cascades in which JAKkinases, particularly JAK3, play a role. Such JAK-dependent signaltransduction cascades include, but are not limited to, the signalingcascades of cytokine receptors that involve the common gamma chain, suchas, for example, the IL-4, IL-7, IL-5, IL-9, IL-15 and IL-21, or IL-2,IL-4, IL-7, IL-9, IL-15 and IL-21 receptor signaling cascades. Thecompounds may also be used in vitro or in vivo to regulate, and inparticular inhibit, cellular or biological responses affected by suchJAK-dependent signal transduction cascades. Such cellular or biologicalresponses include, but are not limited to, IL-4/ramos CD23 upregulation,IL-2 mediated T-cell proliferation, etc. Importantly, the compounds maybe used to inhibit JAK kinases in vivo as a therapeutic approach towardsthe treatment or prevention of diseases mediated, either wholly or inpart, by a JAK kinase activity. Such diseases are referred to as “JAKkinase mediated diseases.”

Compounds I and II are inhibitors of Syk kinases. As a consequence, thecompounds may be used in a variety of in vitro, in vivo and ex vivocontexts to regulate or inhibit Syk kinase activity, signaling cascadesin which Syk kinases play a role, and the biological responses effectedby such signaling cascades. For example, in one embodiment, thecompounds may be used to inhibit Syk kinase, either in vitro or in vivo,in virtually any cell type expressing the Syk kinase.

While not wishing to be bound by theory, it is believed that compoundsdescribed herein are effective treatments of vitiligo due, at least inpart, to their JAK and/or Syk inhibitory activity. In one embodiment,compound I and/or II are employed as salt forms. In a particularembodiment, compound II is used as a salt form. In one embodiment, thesalt of compound II is selected from the sodium salt, the potassiumsalt, the calcium salt, the arginine salt and the choline salt.

Co-Administration

When used to treat vitiligo patches of the skin and/or mucous membranes,compounds I and II may be administered singly, as mixtures and/or incombination with other agents useful for activating a prodrug ortreating diseases and/or disorders of the skin. Compounds I and/or IImay be administered in mixture or in combination with agents useful totreat other disorders or maladies, such as steroids, membranestabilizers, 5-lipoxygenase (5LO) inhibitors, leukotriene synthesis andreceptor inhibitors, inhibitors of IgE isotype switching or IgEsynthesis, IgG isotype switching or IgG synthesis, β-agonists, tryptaseinhibitors, aspirin, cyclooxygenase (COX) inhibitors, methotrexate,anti-TNF drugs, rituxan, PD4 inhibitors, p38 inhibitors, PDE4inhibitors, and antihistamines, to name a few. Compounds I and II may beadministered per se, in the form of prodrugs, salts, solvates, orN-oxides, or as pharmaceutical compositions, comprising the activecompound and/or prodrug.

The pharmaceutical compositions disclosed herein can be co-administered(concurrently or sequentially) with a variety of other treatmentsapplied to the skin, for example antibacterials (such as Bactroban® orCleocin®); antipsoriasis medications (such as Micanol®); antifungalagents (such as Lamisil®, Lotrimin®, and Nizoral®); acne treatments(such as benzoyl peroxide topical preparations); treatments forseborrheic dermatitis (such as coal tar); corticosteroids; retinoids(such as Retin-A and Tazorac®) which are gels or creams derived fromvitamin A that are used to treat conditions including acne; and warttreatments (such as salicylic acid), topical immunomodulators (such astacrolimus or pimecrolimus), or psoralen (a UV sensitizer used inconjunction with phototherapy). Any of these agents can be provided intopical or cosmetic formulations, for example in lotions, ointments,creams, gels, soaps, shampoos, or adherent applicators such as patches.

The pharmaceutical compositions disclosed herein can also beco-administered (concurrently or sequentially) with a variety of othertreatments that are not applied to the skin, for example treatments thatare administered systemically, such as orally or parenterally. Examplesof such systemic treatments include corticosteroids (such asPrednisone), antibiotics (such as erythromycin, tetracycline, anddicloxacillin), antifungal agents (such as ketoconazole and Diflucan®),antiviral agents (such as Valtrex®, acyclovir, and Famvir®),corticosteroids, immunosuppressants (such as Cytoxan®, azathioprine,methotrexate, mycophenolate), biologics (such as Rituxan®, Enbrel®,Humira®, Remicade®, Stelara®, and Amevive®), and/or thyroid hormonereplacement.

Other therapies that can be used in combination with compounds I and/orII include, for example, mercaptopurine, corticosteroids such asprednisone, methylprednisolone and prednisolone, alkylating agents suchas cyclophosphamide, calcineurin inhibitors such as cyclosporine,sirolimus and tacrolimus, inhibitors of inosine monophosphatedehydrogenase (IMPDH) such as mycophenolate, mycophenolate mofetil,azathioprine, various antibodies, for example, antilymphocyte globulin(ALG), antithymocyte globulin (ATG), monoclonal anti-T-cell antibodies(OKT3), and irradiation. These various agents can be used in accordancewith their standard or common dosages, as specified in the prescribinginformation accompanying commercially available forms of the drugs (seealso, the prescribing information in the 2006 Edition of The Physician'sDesk Reference). Azathioprine is currently available from SalixPharmaceuticals, Inc. under the brand name Azasan®; mercaptopurine iscurrently available from Gate Pharmaceuticals, Inc. under the brand namePurinethol®; prednisone and prednisolone are currently available fromRoxane Laboratories, Inc.; methyl prednisolone is currently availablefrom Pfizer; sirolimus (rapamycin) is currently available fromWyeth-Ayerst under the brand name Rapamune®; tacrolimus is currentlyavailable from Fujisawa under the brand name Prograf®; cyclosporine iscurrent available from Novartis under the brand name Sandimmune® andAbbott under the brand name Gengraf®; IMPDH inhibitors such asmycophenolate mofetil and mycophenolic acid are currently available fromRoche under the brand name Cellcept® and Novartis under the brand nameMyfortic®; azathioprine is currently available from Glaxo Smith Klineunder the brand name Imuran®; and antibodies are currently availablefrom Ortho Biotech under the brand name Orthoclone®, Novartis under thebrand name Simulect® (basiliximab) and Roche under the brand nameZenapax® (daclizumab).

In one embodiment, the compound of formula I and/or II, or thepharmaceutically acceptable salt form thereof, is administered either incombination or adjunctively with systemic phototherapy (e.g.,narrow-band UV-B phototherapy, 310-315 nm), psoralen photochemotherapy(PUVA: psoralens (e.g., 5-methoxypsoralen, 8-methoxypsoralen (0.1-0.3%),trimethylpsoralen) combined with UV-A light), excimer laser (308 nm)therapy. In some examples, topical tacrolimus (0.03-0.1%) ointment iscombined with excimer laser therapy. Pimecrolimus (1%) cream may becombined with narrow-band UV-B treatment for vitiligo of the face.Vitamin D analogs (e.g., calcipotriol, tacalcitol) may be combined withnarrow-band UV-B or PUVA treatment.

In another embodiment, compound I and/or compound II is administeredeither in combination or adjunctively with the therapies above and/orwith a therapy for another disease. For example, the compound may becombined with thyroid hormone replacement therapy or withanti-inflammatory or immunomodulatory therapies.

In one embodiment, compound I and/or compound II is administered eitherin combination or adjunctively with an ophthalmic formulation of a drugsuch as an antihistamine, an antibiotic, an anti-inflammatory, anantiviral or a glaucoma medication for treating cases of vitiligo thatprimarily affect the eye or skin around the eye (such as the eyelids),and may be administered to or around the eye, for example in drops orointments. When preparing these combination formulations, compound Iand/or compound II may be combined with ophthalmic antibiotics (such assulfacetamide, erythromycin, gentamicin, tobramycin, ciprofloxacin orofloxacin); ophthalmic corticosteroids (such as prednisolone,fluorometholone or dexamethasone; ophthalmic non-steroidalanti-inflammatories (such as ibuprofen, diclofenac, ketorolac orflurbiprofen); ophthalmic antihistamines (such as livostin, patanol,cromolyn, alomide, or pheniramine); ophthalmic antiviral eye medications(such as triflurthymidine, adenine, arabinoside or idoxuridine);ophthalmic glaucoma medications (for example beta-blockers such astimolol, metipranolol, carteolol, betaxolol or levobunolol); ophthalmicprostaglandin analogues (such as latanoprost); ophthalmic cholinergicagonists (such as pilocarpine or carbachol); ophthalmic alpha agonistssuch as bromonidine or iopidine; ophthalmic carbonic anhydraseinhibitors (such as dorzolamide); and ophthalmic adenergic agonists(such as epinephrine or dipivefrin).

Additional compounds that can be substituted for compounds I and II inthe disclosed methods are specifically contemplated herein and aredescribed in Argade et al. U.S. Pat. No. 7,491,732, issued Feb. 17, 2009and U.S. Patent Application Publication No. 2007/0203161, published Aug.30, 2007, both of which are incorporated herein by reference.

V. Examples Example 1 Compound Synthesis

Compounds I and II, as well as salts III-VII, are synthesized asdescribed below or by analogy to the syntheses described below.Alternative syntheses would be appreciated by one of ordinary skill inthe art.

I:N2-(3-Aminosulfonyl-4-methylphenyl)-5-fluoro-N4-[4-(prop-2-ynyloxy)phenyl]-2,4-pyrimidinediamine

4-Nitrophenol (1.00 g, 7.19 mmol), propargyl bromide (80 wt % intoluene; 0.788 mL, 7.09 mmol), and K₂CO₃ (1.08 g, 7.84 mmol) werecombined and stirred in acetone (16.0 mL) at 60° C. for 18 h. Thereaction mixture was cooled to room temperature and diluted with water(200 mL). 4-(prop-2-ynyloxy)nitrobenzene was isolated as a white solidby suction filtration (1.12 g). ¹H NMR (CDCl₃): δ 8.22 (d, J=9.0 Hz,2H), 7.05 (d, J=9.0 Hz, 2H), 4.80 (d, J=2.4 Hz, 2H), 2.59 (t, J=2.4 Hz,1H).

4-(Prop-2-ynyloxy)nitrobenzene (0.910 g, 5.13 mmol), iron (1.42 g, 25.3mmol), and NH₄Cl (0.719 g, 12.8 mmol) were vigorously stirred inEtOH/water (1:1, 55 mL) at 70° C. for 15 minutes. The reaction mixturewas filtered hot through diatomaceous earth and concentrated in vacuo.The residue was suspended in 10% 2N ammoniacal methanol indichloromethane, sonicated, and filtered through diatomaceous earth.Concentration gave 4-(prop-2-ynyloxy)aniline as an oil which was usedwithout further purification. ¹H NMR (CDCl₃): δ 6.82 (d, J=8.7 Hz, 2H),6.64 (d, J=8.7 Hz, 2H), 4.61 (d, J=2.4 Hz, 2H), 2.50 (t, J=2.4 Hz, 1H).

4-(prop-2-ynyloxy)aniline (0.750 g, 5.10 mmol) and2,4-dichloro-5-fluoropyrimidine (1.27 g, 0.760 mmol, commerciallyavailable from Sigma-Aldrich of Milwaukee, Wis., USA) were stirred inMeOH/water (4:1, 35 mL) at room temperature for 18 h. The reactionmixture was diluted with EtOAc (200 mL) and washed with 1N HCl (50 mL)and brine (50 mL). The organic layer was dried (MgSO₄), filtered andconcentrated in vacuo. The residue was purified by column chromatography(silica gel, hexanes ramped to EtOAc:hexanes (1:10)) to provide2-chloro-5-fluoro-N4-[4-(prop-2-ynyloxy)phenyl]-4-pyrimidineamine as alight brown solid (0.514 g). ¹H NMR (CDCl₃): δ 8.03 (d, J=2.7 Hz, 1H),7.53 (d, J=8.7 Hz, 2H), 7.02 (d, J=8.7 Hz, 2H), 6.86 (s, 1H), 4.71 (d,J=2.4 Hz, 2H), 2.55 (t, J=2.4 Hz, 1H); LCMS: purity: 99%; MS (m/e): 279(MH⁺).

2-Chloro-5-fluoro-N4-[4-(prop-2-ynyloxy)phenyl]-4-pyrimidineamine (0.514g, 1.85 mmol), 3-(aminosulfonyl)-4-methylaniline (0.689 g, 3.70 mmol,made by reduction of commercially available2-methyl-5-nitrobenzenesulfonamide or synthesized as described below),and trifluoroacetic acid (0.186 mL, 2.41 mmol) were combined with iPrOH(6.0 mL) in a sealed vial and heated at 100° C. for 3 h. The reactionmixture was cooled to room temperature and diluted with 1N HCl (80 mL).N2-(3-Aminosulfonyl-4-methylphenyl)-5-fluoro-N4-[4-(prop-2-ynyloxy)phenyl]-2,4-pyrimidinediamine(I) was isolated as a white solid by suction filtration (0.703 g). ¹HNMR (DMSO-d₆): δ 10.08 (bs, 2H), 8.19 (d, J=4.5 Hz, 1H), 7.89 (s, 1H),7.74 (dd, J=2.4 and 8.4 Hz, 1H), 7.58 (d, J=8.7 Hz, 2H), 7.32 (bs, 2H),7.23 (d, J=8.4 Hz, 1H), 6.97 (d, J=8.4 Hz, 2H), 4.79 (d, J=2.1 Hz, 2H),3.59-3.55 (m, 1H), 2.53 (s, 3H); LCMS: purity: 97%; MS (m/e): 428 (MH⁺).

II:5-fluoro-N2-(4-methyl-3-propionylaminosulfonylphenyl)-N4-[4-(prop-2-ynyloxy)phenyl]-2,4-pyrimidinediamine

N2-(3-Aminosulfonyl-4-methylphenyl)-5-fluoro-N4-[4-(prop-2-ynyloxy)phenyl]-2,4-pyrimidinediamine,I, (0.200 g, 0.467 mmol), DMAP (40 mg, 0.33 mmol)) and triethylamine(0.118 mL, 0.847 mmol) were stirred in THF (6.0 mL). Propionic anhydride(0.180 mL, 1.40 mmol) was added to the solution drop wise. The reactionmixture was stirred at room temperature overnight. The solution wasdiluted with ethyl acetate (50 mL) and washed with water (5×25 mL) andbrine (10 mL). The organic layer was dried (MgSO₄), filtered, andevaporated. The residue was suspended in ethyl acetate (25 mL),sonicated and the solid collected by filtration to give5-fluoro-N2-(4-methyl-3-propionylaminosulfonylphenyl)-N4-[4-(prop-2-ynyloxy)phenyl]-2,4-pyrimidinediamine,II, (0.20 g). ¹H NMR (DMSO-d₆): δ 12.01 (s, 1H), 9.44 (s, 1H), 9.26 (s,1H), 8.16 (d, J=2.4 Hz, 1H), 8.06 (dd, J=0.3 and 3.3 Hz, 1H), 8.00 (dd,J=2.1 and 7.8 Hz, 1H), 7.69 (d, J=8.7 Hz, 2H), 7.19 (d, J=8.4 Hz, 1H),6.95 (d, J=8.7 Hz, 2H), 4.77 (d, J=2.1 Hz, 2H), 3.56 (t, J=2.1 Hz, 1H),2.49 (s, 3H), 2.24 (q, J=7.2 Hz, 2H), 0.89 (t, J=7.2 Hz, 3H); LCMS:purity: 98%; MS (m/e): 484 (MH+).

III:5-fluoro-N2-(4-methyl-3-propionylaminosulfonylphenyl)-N4-[4-(prop-2-ynyloxy)phenyl]-2,4-pyrimidinediaminemono-sodium salt

5-Fluoro-N2-(4-methyl-3-propionylaminosulfonylphenyl)-N4-[4-(prop-2-ynyloxy)phenyl]-2,4-pyrimidinediamine,II, (0.125 g, 0.258 mmol) was suspended in acetonitrile (1.5 mL) andwater (1.5 mL) and cooled in an ice bath. A solution of 1N NaOH aq.(0.260 mL) was added drop wise. The reaction mixture was stirred untilit became clear, filtered through glass wool, and lyophilized to givethe sodium salt of II. ¹H NMR (DMSO-d₆): δ 9.17 (bs, 2H), 8.01 (d, J=3.6Hz, 1H), 7.89 (s, 1H), 7.78-7.69 (m, 3H), 6.99-6.92 (m, 3H), 4.76 (d,J=2.1 Hz, 1H), 2.43 (s, 3H), 1.95 (q, J=7.2 Hz, 2H), 0.86 (t, J=7.2 Hz,3H); LCMS: purity: 98%; MS (m/e): 484 (MH⁺).

The following compounds were made in a similar fashion to those above.

IV:5-Fluoro-N2-[4-methyl-3-(N-propionylaminosulfonyl)phenyl]-N4-[4-(2-propynyloxy)phenyl]-2,4-pyrimidinediaminePotassium Salt

¹H NMR (DMSO-d₆): δ 9.16 (s, 1H), 9.14 (s, 1H), 8.01 (d, J=3.6 Hz, 1H),7.85 (d, J=2.1 Hz, 1H), 7.75-7.70 (m, 3H), 6.97-6.92 (m, 3H), 4.76 (d,J=1.8 Hz, 2H), 3.55 (t, J=2.4 Hz, 1H), 2.42 (s, 3H), 1.91 (q, J=7.5 Hz,2H), 0.85 (t, J=7.5 Hz, 3H); LCMS: purity: 97%; MS (m/z): 484 (parent,MH⁺).

V:5-Fluoro-N2-[4-methyl-3-(N-propionylaminosulfonyl)phenyl]-N4-[4-(2-propynyloxy)phenyl]-2,4-pyrimidinediamineCalcium Salt

¹H NMR (DMSO-d₆): δ 9.16 (s, 2H), 8.00 (d, J=3.6 Hz, 1H), 7.88 (d, J=1.8Hz, 1H), 7.75-7.69 (m, 3H), 6.97-6.92 (m, 3H), 4.76 (d, J=1.8 Hz, 2H),3.55 (t, J=2.1 Hz, 1H), 2.43 (s, 3H), 1.94 (q, J=7.5 Hz, 2H), 0.87 (t,J=7.5 Hz, 3H); LCMS: purity: 98%; MS (m/z): 484 (parent, MH⁺).

VI:5-Fluoro-N2-[4-methyl-3-(N-propionylaminosulfonyl)phenyl]-N4-[4-(2-propynyloxy)phenyl]-2,4-pyrimidinediamineArginine Salt

¹H NMR (D₂O): δ 7.61 (d, J=3.9 Hz, 1H), 7.57-7.55 (m, 1H), 7.36-7.31 (m,1H), 7.12 (d, J=8.7 Hz, 2H), 6.88 (d, J=8.7 Hz, 1H), 6.72 (d, J=9.0 Hz,2H), 4.77-4.75 (m, 2H), 3.60 (t, J=6.0 Hz, 1H), 3.09 (t, J=6.9 Hz, 2H),2.84-2.81 (m, 1H), 2.35 (s, 3H), 2.03 (q, J=5.7 Hz, 2H), 1.80-1.72 (m,2H), 1.61-1.48 (m, 2H), 0.855 (t, J=7.5 Hz, 3H); LCMS: purity: 98%; MS(m/z): 484 (parent, MH⁺).

VII:5-Fluoro-N2-[4-methyl-3-(N-propionylaminosulfonyl)phenyl]-N4-[4-(2-propynyloxy)phenyl]-2,4-pyrimidinediamineCholine Salt

¹H NMR (DMSO-d₆): δ 9.16 (s, 2H), 8.00 (d, J=3.6 Hz, 1H), 7.85 (d, J=1.8Hz, 1H), 7.75-7.69 (m, 3H), 6.97-6.90 (m, 3H), 5.27 (t, J=4.8 Hz, 1H),4.76 (d, J=1.8 Hz, 2H), 3.86-3.77 (m, 2H), 3.56-3.54 (m, 1H), 3.40-3.54(m, 2H), 3.08 (s, 9H), 2.42 (s, 3H); LCMS: purity: 99%; MS (m/z): 484(parent, MH⁺).

5-amino-2-methylbenzenesulfonamide

4-methylnitrobenzene (20 mmol) is treated at 0° C. with chlorosulfonicacid (5.29 mL, 80 mmol) and then, after bringing the homogeneoussolution to room temperature, it was stirred at 110° C. for 24 hours.The resulting slurry was then poured over ice water (100 gm), extractedwith diethyl ether (3×75 mL), and the organic phase washed with water(75 mL), then dried over anhydrous sodium sulfate. The solvent was thenremoved under reduced pressure to afford the crude sulfonyl chloridewhich was taken up in ethyl acetate and stirred with ammonium hydroxideovernight at room temperature. After the ethyl acetate layer wasseparated, the aqueous layer was extracted with ethyl acetate. Theorganic layers were combined, dried over anhydrous sodium sulfate andthe solvent was removed under reduced pressure. The oil obtained waspurified by column chromatography (silica gel, hexanes then 10%, 20%, upto 50% ethyl acetate in hexanes to afford3-aminosulfonyl-4-methylnitrobenzene, LCMS: purity: 95%; MS (m/e): 217(MH+).

To a solution of 3-aminosulfonyl-4-methylnitrobenzene in dichloromethaneand methanol was added 10% Pd/C and the mixture shaken under a hydrogenatmosphere at 50 psi for 15 minutes. The mixture was filtered throughdiatomaceous earth and the filter cake was washed with methanol. Thecombined organic solvents were concentrated under reduced pressure togive crude product, which was further purified by flash columnchromatography (ethyl acetate:hexanes 1:1) to give3-aminosulfonyl-4-methylaniline, LCMS: purity: 87%; MS (m/e): 187 (MH+).

Example 2 Assay for Ramos B-Cell Line Stimulated with IL-4

One means of assaying for JAK inhibition is detection of the effect ofcompounds I and II on the upregulation of downstream gene products. Inthe Ramos/IL4 assay, B-cells are stimulated with the cytokineInterleukin-4 (IL-4) leading to the activation of the JAK/Stat pathwaythrough phosphorylation of the JAK family kinases, JAK1 and JAK3, whichin turn phosphorylate and activate the transcription factor Stat-6. Oneof the genes upregulated by activated Stat-6 is the low affinity IgEreceptor, CD23. To study the effect of inhibitors (for example, the2,4-substituted pyrimidinediamine compounds described herein) on theJAK1 and JAK3 kinases, human Ramos B cells are stimulated with humanIL-4. Twenty to 24 hours post stimulation, cells are stained forupregulation of CD23 and analyzed using flow cytometry (FACS). Areduction of the amount of CD23 present compared to control conditionsindicates the test compound actively inhibits the JAK kinase pathway. Anexemplary assay of this type is described in greater detail below.

B-cells stimulated with cytokine Interleukin-4 (IL-4) activate theJAK/Stat pathway through phosphorylation of the JAK family kinases,JAK-1 and JAK-3, which in turn phosphorylate and activate thetranscription factor Stat-6. One of the genes upregulated by activatedStat-6 is the low affinity IgE receptor, CD23. To study the effect ofinhibitors on the JAK family kinases, human Ramos B cells are stimulatedwith human IL-4.

The Ramos B-cell line was acquired from ATCC (ATCC Catalog No.CRL-1596). The cells were cultured in RPMI 1640 (Cellgro, MediaTech,Inc., Herndon, Va., Cat No. 10-040-CM) with 10% fetal bovine serum(FBS), heat inactivated (JRH Biosciences, Inc, Lenexa, Kans., Cat No.12106-500M) according to ATCC propagation protocol. Cells weremaintained at a density of 3.5×10⁵. The day before the experiment, RamosB-cells were diluted to 3.5×10⁵ cells/mL to ensure that they were in alogarithmic growth phase.

Cells were spun down and suspended in RPMI with 5% serum. 5×10⁴ cellswere used per point in a 96-well tissue culture plate. Cells werepre-incubated with compound or DMSO (Sigma-Aldrich, St. Louis, Mo., CatNo. D2650) vehicle control for 1 hour in a 37° C. incubator. Cells werethen stimulated with IL-4 (Peprotech Inc., Rocky Hill, N.J., Cat No.200-04) for a final concentration of 50 units/mL for 20-24 hours. Cellswere then spun down and stained with anti-CD23-PE(BD Pharmingen, SanDiego, Calif., Cat No. 555711) and analyzed by FACS. Detection wasperformed using a BD LSR I System Flow Cytometer, purchased from BectonDickinson Biosciences of San Jose, Calif. The IC₅₀ calculated based onthe results of this assay are provided in Table 1.

Example 3 Primary Human T-Cell Proliferation Assay Stimulated with IL-2

The JAK activity of the compounds described herein may further becharacterized by assaying the effect of compounds I and II describedherein on the proliferative response of primary human T-cells. In thisassay, primary human T-cells derived from peripheral blood andpre-activated through stimulation of the T-cell receptor and CD28,proliferate in culture in response to the cytokine Interleukin-2 (IL-2).This proliferative response is dependent on the activation of JAK1 andJAK3 tyrosine kinases, which phosphorylate and activate thetranscription factor Stat-5. The primary human T-cells are incubatedwith compounds I and II in the presence of IL-2 for 72 hours and at theassay endpoint intracellular ATP concentrations are measured to assesscell viability. A reduction in cell proliferation compared to controlconditions is indicative of inhibition of the JAK kinase pathway. Anexemplary assay of this type is described in greater detail below.

Primary human T-cells derived from peripheral blood and pre-activatedthrough stimulation of the T-cell receptor and CD28, proliferate invitro in response to the cytokine Interleukin-2 (IL-2). Thisproliferative response is dependent on the activation of JAK-1 and JAK-3tyrosine kinases, which phosphorylate and activate the transcriptionfactor Stat-5.

Human primary T cells were prepared as follows. Whole blood was obtainedfrom a healthy volunteer, mixed 1:1 with PBS, layered on to FicollHypaque (Amersham Pharmacia Biotech, Piscataway, N.J., Catalog#17-1440-03) in 2:1 blood/PBS:ficoll ratio and centrifuged for 30 min at4° C. at 1750 rpm. The lymphocytes at the serum: ficoll interface wererecovered and washed twice with 5 volumes of PBS. The cells wereresuspended in Yssel's medium (Gemini Bio-products, Woodland, Calif.,Catalog #400-103) containing 40 U/mL recombinant IL2 (R and D Systems,Minneapolis, Minn., Catalog #202-IL (20 μg)) and seeded into a flaskpre-coated with 1 μg/mL anti-CD3 (BD Pharmingen, San Diego, Calif.,Catalog #555336) and 5 μg/mL anti-CD28 (Immunotech, Beckman Coulter ofBrea Calif., Catalog #IM1376). The primary T-cells were stimulated for3-4 days, then transferred to a fresh flask and maintained in RPMI with10% FBS and 40 U/mL IL-2.

Primary T-cells were washed twice with PBS to remove the IL-2 andresuspended in Yssel's medium at 2×10⁶ cells/mL. 50 μL of cellsuspension containing 80 U/mL IL-2 was added to each well of a flatbottom 96 well black plate. For the unstimulated control, IL-2 wasomitted from the last column on the plate. Compounds were seriallydiluted in dimethyl sulfoxide (DMSO, 99.7% pure, cell culture tested,Sigma-Aldrich, St. Louis, Mo., Catalog No. D2650) from 5 mM in 3-folddilutions, and then diluted 1:250 in Yssel's medium. 50 μL of 2×compound was added per well in duplicate and the cells were allowed toproliferate for 72 hours at 37° C.

Proliferation was measured using CellTiter-Glo® Luminescent CellViability Assay (Promega), which determines the number of viable cellsin culture based on quantitation of the ATP present, as an indicator ofmetabolically active cells. The substrate was thawed and allowed to cometo room temperature. After mixing the Cell Titer-Glo reagent and diluenttogether, 100 μL was added to each well. The plates were mixed on anorbital shaker for two minutes to induce lysis and incubated at roomtemperature for an additional ten minutes to allow the signal toequilibrate. Detection was performed using a Wallac Victor2 1420multilabel counter purchased from Perkin Elmer, Shelton, Conn. The IC₅₀calculated based on the results of this assay are provided in Table 1.

Example 4 A549 Epithelial Line Stimulated with IFNγ

The JAK activity of the compounds described herein may also becharacterized by assaying the effect of compounds I and II describedherein on A549 lung epithelial cells and U937 cells. A549 lungepithelial cells and U937 cells up-regulate ICAM-1 (CD54) surfaceexpression in response to a variety of different stimuli. Therefore,using ICAM-1 expression as readout, test compound effects on differentsignaling pathways can be assessed in the same cell type. Stimulationwith IL-1β through the IL-1β receptor activates the TRAF6/NFκB pathwayresulting in up-regulation of ICAM-1. IFNγ induces ICAM-1 up-regulationthrough activation of the JAK1/JAK2 pathway. The up-regulation of ICAM-1can be quantified by flow cytometry across a compound dose curve andEC₅₀ values are calculated. Exemplary assays of this type are describedin greater detail below and in Example 6.

A549 lung epithelial cells up-regulate ICAM-1 (CD54) surface expressionin response to a variety of different stimuli. Therefore, using ICAM-1expression as readout, compound effects on different signaling pathwayscan be assessed in the same cell type. IFNγ up-regulates ICAM-1 throughactivation of the JAK/Stat pathway. In this example, the up-regulationof ICAM-1 by IFNγ was assessed.

The A549 lung epithelial carcinoma cell line originated from theAmerican Type Culture Collection. Routine culturing was with F12K media(Mediatech Inc., Lenexa, Kans., Cat. No. 10-025-CV) with 10% fetalbovine serum, 100 I.U. penicillin and 100 ng/mL streptomycin (completeF12k media). Cells were incubated in a humidified atmosphere of 5% CO₂at 37° C. Prior to use in the assay, A549 cells were washed with PBS andtrypsinized (Mediatech Inc., Cat. No. 25-052-CI) to lift the cells. Thetrypsin cell suspension was neutralized with complete F12K media andcentrifuged to pellet the cells. The cell pellet was resuspended incomplete F12K media at a concentration of 2.0×10⁵/mL. Cells were seededat 20,000 per well, 100 μL total volume, in a flat bottom tissue cultureplate and allowed to adhere overnight.

On day two, A549 cells were pre-incubated with a 2,4-substitutedpyrimidinediamine test compound or DMSO (control) (Sigma-Aldrich, St.Louis, Mo., Catalog No. D2650) for 1 hour. The cells were thenstimulated with IFNγ (75 ng/mL) (Peprotech Inc., Rocky Hill, N.J., Cat.No. 300-02) and allowed to incubate for 24 hours. The final testcompound dose range was 30 μM to 14 nM in 200 μL F12K media containing5% FBS, 0.3% DMSO.

On day three, the cell media was removed and the cells were washed with200 μL PBS (phosphate buffered saline). Each well was trypsinized todissociate the cells, then neutralized by addition of 200 μL completeF12K media. Cells were pelleted and stained with an APC conjugated mouseanti-human ICAM-1 (CD54) (BD Pharmingen, San Diego, Calif., Catalog#559771) antibody for 20 minutes at 4° C. Cells were washed with icecold FACS buffer (PBS+2% FBS) and surface ICAM-1 expression was analyzedby flow cytometry. Detection was performed using a BD LSR I System FlowCytometer, purchased from BD Biosciences of San Jose, Calif. Events weregated for live scatter and the geometric mean was calculated(Becton-Dickinson CellQuest software version 3.3, Franklin Lakes, N.J.).Geometric means were plotted against the compound concentration togenerate a dose response curve. The IC₅₀ calculated based on the resultsof this assay are provided in Table 1.

Example 5 U937 IFNγICAM1 FACS Assay

U937 human monocytic cells up-regulate ICAM-1 (CD54) surface expressionin response to a variety of different stimuli. Therefore, using ICAM-1expression as readout, compound effects on different signaling pathwayscan be assessed in the same cell type. IFNγ up-regulates ICAM-1 throughactivation of the JAK/Stat pathway. In this example, the up-regulationof ICAM-1 by IFNγ was assessed.

The U937 human monocytic cell line was obtained from ATCC of RockvilleMd., catalog number CRL-1593.2, and cultured in RPM1-1640 mediumcontaining 10% (v/v) FCS. U937 cells were grown in 10% RPMI. The cellswere then plated at a concentration of 100,000 cells per 160 μL in 96well flat bottom plates. The test compounds were then diluted asfollows: 10 mM test compound was diluted 1:5 in DMSO (3 μL 10 mM testcompound in 12 μL DMSO), followed by a 1:3 serial dilution of testcompound in DMSO (6 μL test compound serially diluted into 12 μL DMSO togive 3-fold dilutions). Then 4 μL of test compound was transferred to 76μL of 10% RPMI resulting in a 10× solution (100 μM test compound, 5%DMSO). For control wells, 4 μL of DMSO was diluted into 76 μL 10% RPMI.

The assay was performed in duplicate with 8 points (8 3-fold dilutionconcentrations from 10 μl) and with 4 wells of DMSO only (control wells)under stimulated conditions and 4 wells of DMSO only under unstimulatedconditions.

The diluted compound plate was mixed 2× using a multimek (BeckmanCoulter of Brea, Calif.) and then 20 μL of the diluted compounds wastransferred to the 96-well plate containing 160 μL of cells, which werethen mixed again twice at low speeds. The cells and compounds were thenpre-incubated for 30 minutes at 37° C. with 5% CO₂.

The 10× stimulation mix was made by preparing a 100 ng/mL solution ofhuman IFNγ in 10% RPMI. The cells and compound were then stimulated with20 μL of IFNγ stimulation mix to give a final concentration of 10 ng/mLIFNγ, 10 μM test compound, and 0.5% DMSO. The cells were kept underconditions for stimulation for 18-24 hours at 37° C. with 5% CO₂.

The cells were transferred to a 96 well round bottom plate for stainingand then kept on ice for the duration of the staining procedure. Cellswere spun down at 1000 rpm for 5 minutes at 4° C., following which thesupernatant was removed. Following removal of the supernatant, 1 μL APCconjugated mouse anti-human ICAM-1 antibody was added per 100 μL FACSbuffer. The cells were then incubated on ice in the dark for 30 minutes.Following incubation, 150 μL of FACS buffer was added and the cells werecentrifuged at 1000 rpm for 5 minutes at 4° C., following which thesupernatant was removed. After removal of the supernatant, 200 μL ofFACS buffer was added and the cells were resuspended. After suspension,the cells were centrifuged at 1000 rpm for 5 min at 4° C. Supernatantwas then removed prior to resuspension of the cells in 150 μL FACSbuffer.

Detection was performed using a BD LSR I System Flow Cytometer,purchased from BD Biosciences of San Jose, Calif. The live cells weregated for live scatter and the geometric mean of ICAM-APC was measured(Becton-Dickinson CellQuest software version 3.3, Franklin Lakes, N.J.).Both % live cells and ICAM-1 expression was analyzed. The assays for thetest compounds were carried out in parallel with a control compound ofknown activity. The EC₅₀ for the control compound is typically 40-100nM. The IC₅₀ calculated based on the results of this assay are providedin Table 1.

TABLE 1 Compound Example 2 Example 3 Example 4 Example 5 I 0.056 0.18111.338 0.565 II 9.655 III 3.972 IV 2.318 5.560 V 0.373 25.126 VI 0.1040.262 4.973 0.424 VII 0.022 0.053 0.140

Example 6 Pharmaceutical Formulations

This example describes pharmaceutical formulations containing compound Ior II (which will be understood to also include salts thereof). Suchformulations are prepared as known to those of skill in the art andadditional formulations will be readily apparent to those of skill inthe art upon consideration of this Example and additional disclosureherein.

TABLE 2 Formulation No. Formulation Components 1 50 mM pH 7.4 phosphatebuffer, 0.05% Tween ® 80, 0.5% NaCl 2 50 mM pH 7.4 phosphate buffer,0.36% HPMC, 0.2% glycerin, 1% PEG400, 0.35% NaCl 3 5 mM pH 7.4 phosphatebuffer, 0.36% HPMC, 0.2% glycerin, 1% PEG400, 5% Cremophor ® ELP, 4.3%mannitol 4 10 mM pH 5.8 citrate buffer, 4.2% mannitol 5 10 mM pH 5.8citrate buffer, 4.2% mannitol, 0.36% HPMC, 0.2% glycerin 6 0.3%tyloxapol, 0.5% Carbopol ® 974P, 2.25% mannitol, 50 mM pH 6.5 phosphatebuffer, 230 mOsm/kg 7 0.3% tyloxapol, 0.1% Carbopol ® 974P, 2.25%mannitol, 50 mM pH 6.5 phosphate buffer, 230 mOsm/kg

Each of the above formulations, 1-7, are prepared with compound I or IIin three dosage concentrations: 0.001%, 0.003% and 0.01% (w/w). Eachformulation is prepared by adding the specified amount of a tonicityagent (mannitol) to a flask, heating to about 50° C. in about half thefinal volume of the specified buffer (phosphate or citrate). Afterheating, the appropriate amount of compound I or II is added along withthe additional excipients (glycerin and/or PEG400) as indicated.Purified water is added in sufficient quantity. The mixture is stirredto homogeneity (about five minutes) and then filtered through asterilizing filter membrane into a sterile vessel. If necessary, pH isadjusted by addition of 1.0 N NaOH.

Optionally, formulations having a higher concentration of compound I orII (for example, 0.03% w/w) can include a surfactant and optionally astabilizing polymer. With reference to formulations 6 and 7, preferredsurfactants include Triton® X114 and tyloxapol, which are commerciallyavailable from Sigma-Aldrich (of St. Louis, Mo.) and Pressure ChemicalCompany (of Pittsburgh, Pa.), respectively. Preferred stabilizingpolymers include the carbomer Carbopol® 974p (commercially availablefrom Lubrizol, of Wickliffe, Ohio).

Formulations 6 and 7 are prepared by dispersing the carbomer first inthe surfactant containing buffer at 10× of their final concentration(e.g. 3% tyloxapol in 50 mM phosphate buffer at pH 6.5 with 2.5%mannitol and 5% Carbopol® 974p). Either compound I or compound II isthen dispersed in this preconcentrate also at 10× of its finalconcentration. The mixture is homogenized, with final formulation beingobtained by 10× dilution of filtered preconcentrate in a matchingbuffer.

Methods of formulating and testing the drugs for topical application aredescribed, for example, in Remington, The Science and Practice ofPharmacy (21^(st) edition), pages 872-882 (2006). The drug is formulatedfor delivery of drug to a desired depth of the skin surface, whileavoiding unwanted systemic absorption of the drug. Various penetrationenhancers can be added to the composition, such as an alcohol, alkylmethyl sulfoxide, pyrrolidone, laurocapram, dimethyl formamide,tetrahydrofurfuryl alcohol, an amphiphile, or other miscellaneousenhancers such as clofibric acid amide, hexamethylene lauramide,proteolytic enzymes, terpenes or sesquiterpenes. The penetrationenhancers improve drug delivery into the skin.

In one specific example of the formulation, a 60:20:20 ethanol:propyleneglycol:water system is used with sufficient propylene glycol to maintain0.5-2% of the active compound.

Common ingredients which may be used to administer the compound in atopical formulation are vehicles, for example hydrophobic vehicles suchas hydrocarbons, liquid petrolatum (mineral oil, liquid paraffin,paraffin oil), white petrolatum (petroleum jelly, Vaseline®), yellowpetrolatum (petroleum jelly), squalane (perhydrosqualene, spinacane),and silicones; silicones such as liquid polydimethylsiloxanes(dimethicone, silastic, medical grade silicone oil), alcohols such aslauryl alcohols (1-dodecanol, dodecyl alcohols), myristyl alcohols(tetradecanol, tetradecyl alcohols), cetyl alcohols (hexadecanol, ethal,palmityl alcohols), stearyl alcohols (stenol, cetosteryl alcohols),oleyl alcohols (ocenol); sterols such as sterol esters; lanolin such ashydrous wool fat, lanum; anhydrous lanolin (such as wool fat, anhydrouslanum, agnin); semi synthetic lanolins; carboxylic acids such as lauricacid, myristic acid, palmitic acid, stearic acid, oleic acid; esters andpolyesters, such as cholesterol esters (stearate), ethylene glycolmonoesters, propylene glycol monoesters, glyceryl monoesters, glycerylmonostearate, sorbitol monoesters, sorbitan monoesters, sorbitoldiesters, sorbitan polyesters (spans, arlacels), glyceryl tristearate,lard, almond oil, corn oil, castor oil, cottonseed oil, olive oil,soybean oil, hydrogenated oils, sulfated oils, isopropyl myristate,isopropyl palmitate; ethers and polyethers such aspolyethylene-polypropylene glycols (pluronics).

Water-miscible vehicles that may be used as co-solvents include polyolsand polyglycols such as propylene glycol (1,2-propanediol), glycerin(glycerol), liquid polyethylene glycol, solid polyethylene glycol (hardmacrogol, carbowax), 1,2-phenol-hexanetriol, sorbitol solution, estersand polyesters such as polyoxyethylene sorbitain monoesters(stearate-tweens) and polyoxy ethylene sorbitan polyesters (tweens);ethers and polyethers such as polyethylene glycol monocetyl ether(cetomacrogol 1000) and polyethylene-polypropylene glycols (pluronics).

Various structural matrix formers can be added to the composition, forexample hydrocarbons such as white petrolatum (petroleum jelly,Vaseline®), yellow petrolatum (petroleum jelly), paraffin (paraffin wax,hard paraffin), microcrystalline wax, ceresin (mineral wax, purifiedozokerite); silicones such as fumed silica (cab-O-sil), bentonite(colloidal aluminum silicate), and veegum (colloidal magnesium aluminumsilicate); polyols and polyglocols such as solid polyethylene glycol(hard macrogol, carbowax); alcohols such as cetyl alcohols (hexadecanol,ethal, palmityl alcohols), stearyl alcohols (stenol, cetosterylalcohols); sterold and sterol esters such as cholesterol (cholesterin),lanolin, anhydrous lanolin, and semisynthetic lanolin; carboxylic acidssuch as lauric acid, myristic acid, palmitic acid, stearic acid, oleicacid; and esters or polyesters such as bees wax, white bees wax(bleached bees wax), Carnauba wax, myricin, cholesterol esters(stearate), polyoxyethylene sorbitain, lard or hydrogenated oils.

The compositions may further include suspending, jelling or viscosityinducing agents, for example silicones such as fumed silica (cab-O-sil),bentonite (colloidal aluminium silicate), or veegum (colloidal magnesiumaluminium silicate); polycarboxylates, polysulfates or polysaccharidessuch as agar, alginates, carragen, acacia, tragacanth, methylcellulose,carboxy methylcellulose, hydroxy ethyl cellulose, carboxy vinyl polymer,gelatin, pectin, xanthan, polyacrylic acid.

Some embodiments may include a water-in-oil emulsifier such as a sterolor sterol ester, for example cholesterol (cholesterin), lanolin (hydrouswool fat, lanum), anhydrous lanolin (wool fat, anhydrous lanum, agnin),or semi synthetic lanolin; carboxylic acids such as Na+, K+, ethanolaminsalts of lauric acid, myristic acid, palmitic acid, stearic acid, oleicacid, or an ether or polyether such as polyethylene-polypropyleneglycols (pluronics). If an oil-in-water (o/w) emulsifier is desired,examples are esters and polyesters such as polyoxyethylene sorbitainmonoesters (stearate-tweens), polyoxy ethylene esters(stearate-polyethylene glycol monoesters, Myrj), polyoxy ethylenesorbitan polyesters (tweens); ethers and polyethers such as polyethyleneglycol monocetyl ether (cetomacrogol 1000) or polyethylene-polypropyleneglycols (pluronics), and others such as sodium lauryl sulfate, borax(sodium borate), ethanolamine, or triethanolamine.

Suitable surfactants for use in the formulations include, but are notlimited to, nonionic surfactants like Surfactant 190 (dimethiconecopolyol), polysorbate 20 (Tween® 20), polysorbate 40 (Tween® 40),polysorbate 60 (Tween® 60), polysorbate 80 (Tween® 80), lauramide DEA,cocamide DEA, and cocamide MEA, amphoteric surfactants like oleylbetaine and cocamidopropyl betaine (Velvetex® BK-35), and cationicsurfactants like Phospholipid PTC (Cocamidopropyl phosphatidylPG-dimonium chloride). Appropriate combinations or mixtures of suchsurfactants may also be used.

Suitable moisturizers for use in the formulations of the presentinvention include, but are not limited to, lactic acid and other hydroxyacids and their salts, glycerin, propylene glycol, butylene glycol,sodium PCA, Carbowax™ 200, Carbowax™ 400, and Carbowax™ 800. Suitableemollients for use in the formulations of the present invention include,but are not limited to, PPG-15 stearyl ether, lanolin alcohol, lanolin,lanolin derivatives, cholesterol, petrolatum, isostearyl neopentanoate,octyl stearate, mineral oil, isocetyl stearate, Ceraphyl® 424 (myristylmyristate), octyl dodecanol, dimethicone (Dow Corning 200-100 cps),phenyl trimethicone (Dow Corning 556), Dow Corning 1401 (cyclomethiconeand dimethiconol), and cyclomethicone (Dow Corning 344), and Miglyol®840 (manufactured by Huls; propylene glycol dicaprylate/dicaprate). Inaddition, appropriate combinations and mixtures of any of thesemoisturizing agents and emollients may be used in accordance with thepresent disclosure.

The composition may also include preservatives and antimicrobials, suchas benzalkonium chloride, benzoic acid, benzyl alcohol, bronopol,chlorhexidine, chlorocresol, imidazolidinyl urea, paraben esters,phenol, phenoxyethanol, potassium sorbate, or sorbic acid; antioxidantssuch as α-tocopherol, ascorbic acid, ascorbyl palmitate, butylatedhydroxyanisole, sodium ascorbate, sodium metabisulfite; chelating agentssuch as citric acid or edetic acid; buffers such as citric acid andsalts, phosphoric acid and salts, H₃PO₄/NaH₂PO₄, glycine, acetic acid,triethanolamine, or boric acid; humectants such as glycerin (glycerol),propylene glycol (E 1520), glyceryl triacetate (E1518), sorbitol (E420),xylitol and malitol (E965), polydextrose (E1200), quillaia (E999),lactic acid, urea or lithium chloride; and/or a sequestering antioxidantsuch as citric acid and it salts ethylenediaminetetraacetic acid(Versene, EDTA).

A particular embodiment of the topical treatment may be an ointment,which is a semisolid preparation intended for external application tothe skin or mucous membranes. In a specific example, the ointment isbased on petrolatum. The ointment does not contain sufficient water toseparate into a second phase at room temperature. A water-solubleointment may be formulated with polyethylene glycol. Ointments are idealemollients with good skin penetration and adherence to surfaces. Theointment is in a convenient container such as a tube or jars.

Alternatively, the topical dosage form is a cream in which the compoundsare dissolved or suspended in water removable or emollient bases. Thecreams may be either water-in-oil or oil-in-water compositions.Immiscible compounds may be combined by mechanical agitation or heatusing wet gum, dry gum, bottle, and beaker methods. In some embodiments,the cream is an oil-in-water emulsion or aqueous microcrystallinedispersion of long chain fatty acids or alcohols that are water washableand more cosmetically and aesthetically acceptable.

In other embodiments, the active ingredients are provided foradministration in a paste, which can be considered an ointment intowhich a high percentage of insoluble solids have been added, for exampleas much as 50% by weight. The paste is much stiffer than the ointmentdue to the presence of solids, which form a particulate matrix over andabove the ointment structure already present. Ingredients such asstarch, zinc oxide, calcium carbonate, and talc are used as the solidphase. Pastes provide a particularly good protective barrier on skinLike ointment, a paste forms an unbroken, relatively water impermeablefilm on the skin surface; unlike ointment the film is opaque andtherefore an effective sun filter.

In yet other embodiments, the active agent is provided in a gel, jellyor lotion. Gels are semisolid systems consisting of dispersions of smallor large molecules in an aqueous liquid vehicle rendering jelly-likethrough the addition of gelling agent. Among the gelling agents used aresynthetic macromolecules such as carbomer 934, and cellulose derivativessuch as carboxymethylcellulose or hydroxypropylmethyl-cellulose. Gelsare compatible with many substances and may contain penetrationenhancers to improve delivery into the skin. The gels may be eithersingle phase gels in which the macromolecules are uniformly distributedthroughout a liquid with no apparent boundaries between the dispersedmacromolecules and the liquid, or double phase gels in which the gelmass consists of floccules of small distinct particles, often referredto as a magmas. A jelly contains a water-soluble base prepared fromnatural gums such as tragacanth, pectin, alginate, or boroglycerin, orfrom synthetic derivatives of a natural substance such asmethylcellulose or carboxymethylcellulose. A lotion is a clear solutioncontaining 25-50% alcohol, which optionally contains an antiseptic, oremollient. Other optional ingredients that may be added to the lotionare an extract of witchhazel, menthol, glycerin, boric acid, alum, orpotassium oxyquinoline.

In another embodiment the compound is applied in a powder, whichcontains very fine particle sizes that produce large surface area perunit weight to covers a larger surface area of the body and providelight dispersion. Alternatively the compound is applied in a solution,which is a liquid preparations of soluble chemicals dissolved insolvents such as water, alcohol, or propylene glycol. In yet otherexamples, it is an emulsion, which is a two-phase preparation in whichone phase (the dispersed or internal phase) is finely dispersed in theother (continuous or external phase). The dispersed phase can haveeither a hydrophobic-base (oil-in-water) or aqueous base (water-in-oil).Because there are two incompatible phases in close conjunction, theemulsion would typically contain a physical stabilizing system, such asa surfactant (ionic or nonionic), polymer (nonionic polymers,polyelectrolytes, or biopolymers), or mixtures thereof.

For embodiments in which the compound is provided in a suspension, thedosage form contains two phases. The continuous or external phase isgenerally a liquid or semisolid while the dispersed or internal phase ismade up of particulate matter that is essentially insoluble in, butdispersed throughout, the continuous phase. The insoluble matter may beintended for physiologic action, for example by external coating.Although the suspension system may separate on standing, the rate ofsettling may be decreased by varying the formulation to retain asufficiently homogenous composition for at least the period of timenecessary to administer the required dose after shaking its container.

The compound may also be administered in an aerosol, which depends onthe power of compressed or liquefied gas to expel the contents from thecontainer. Propellants in the container are responsible for developingthe proper pressure within the container and it expel the product whenthe valve is opened and aids in the atomization or foam production ofthe products. Topical pharmaceuticals aerosols utilize hydrocarbon(propane, butane, and isobutene) and compressed gases such as nitrogen,carbon dioxide, and nitrous oxide.

Any of these dosage forms can contain separate reservoirs of compoundsI/II and an adjunct agent (such as an agent for activating compound IIto form compound I).

Example 7 Vitiligo Animal Models

This example describes the use of animal models to screen for treatmentsfor vitiligo, including the selection of regimens for treatment,prevention and combination treatments. Animal models are used to testthe claimed compounds, as well as combination formulations, such asthose described herein. In particular examples, topical formulationsthat contain compounds I and/or II are applied to the skin of the animaland the therapeutic response is assessed. After the formulations areadministered to the animal, the skin is examined for evidence ofdecreased number or size of depigmented areas.

The C57BL/6J Ler-vit/vit mouse strain has been beneficial as a vitiligoresearch tool (Lerner et al., J. Invest. Dermatol. 87(3):299-304(September 1986)). This strain arose from the C57BL/6J strain. Thevitiligo mouse has congenital dorsal and ventral white spots as well asreplacement of pigmented hairs by white hairs. The lack of pigment isdue to absence of melanocytes from the epidermis and hair follicles.

Another mouse model is the C57BL/6-mi^(vit)/m^(vit) mouse, which has aslowly progressing retinal degeneration with unevenly pigmented retinalpigment epithelium. See, e.g., Smith et al., Invest. Ophthalmol. & Vis.Sci. 35(10):3625-3632 (September 1994).

An alternative animal model is the Smyth line (SL) chicken developed byDr. J. Robert Smyth, Jr. at the University of Massachusetts, Amherst,Mass. See, e.g., Shi et al., BMC Immunol. 13:18 (April 2012); Stepichevaet al., J. Immunol. 184:83.16 (2010).

In the model, the animal is exposed to the test agent, using differentroutes, dosages and regimens of administration. In particular examples,the drugs disclosed herein are applied topically to the areas that havebeen or will be tape stripped. Alternatively, the test drug isadministered systemically. The drug is administered one or more times atfixed intervals prior to or following tape stripping (for example, dailyfollowing tape stripping or following the appearance of depigmented skinareas or depigmented hair patches). Drug response can be assessed bymeasuring such indicia of disease as the number, surface area or size ofdepigmented areas. Histological analysis of skin specimens is alsoperformed to determine the number of melanocytes within the epidermis.Other histological findings may include an increased number ofLangerhans' cells, epidermal vacuolization, thickening of the basementmembrane, T-cell inflammatory infiltrate, and neural alterations (Monteset al., Int. J. Dermatol., 42(1):57-61 (January 2003).) Deposits ofextracellular granular material and/or foci of vacuolar degeneration ofbasal and parabasal keratinocytes may be observed (Moellmann et al., J.Inv. Dermatol. 79:312-330 (1982).) Inflammatory vitiligo may presentwith psoriasiform hyperplasia, parakeratotic mounds, acanthosis withelongation of rete ridges, and/or dilated vessels (Verma, DermatologyOnline Journal 11(3):13 (2005).)

Example 8 Methods of Treatment and Combination Formulations

Subjects to be treated with the claimed formulations are selected basedon a clinical and/or histopathological presentation of vitiligo.Subjects also may be selected based on an increased risk of developingvitiligo, such as a family history of vitiligo and/or presence ofanother disease associated with increased incidence of vitiligo. Theclaimed compositions are generally applied topically to the depigmentedareas on the skin, although they may also be applied more generally tothe skin or administered systemically. Treatment may be continued for atleast a week, month, or year, and in some subjects treatment may extendover multiple years, the duration of disease, or the lifetime of thesubject.

In particular cases, subjects are selected for concomitant treatmentwith other pharmaceutical or non-pharmaceutical interventions, such assystemic Plaquenil®, topical corticosteroid, phototherapy, psoralenphotochemotherapy, or excimer laser therapy. In other cases thecompounds I and/or II are administered with no other treatment forvitiligo.

In one example, the subject is selected by making a diagnosis ofvitiligo. A therapeutically effective amount of the compound is providedin a topical petrolatum jelly formulation and the formulation is applieddirectly to depigmented areas, such as patches on the face andextremities. The pharmaceutical formulation is applied to thedepigmented patches daily, for example 2-4 times per day for more thanone day, for example at least one week. Topical application of theformulation to the depigmented areas is continued until the areas towhich the formulation is applied show evidence of repigmentation ordisappear, or their progression is delayed or stopped.

In other examples, the therapeutic compound is provided in an effectiveamount in a sunscreen formulation and is applied to the skin prior toexposure to ultraviolet radiation, to protect against exposure toultraviolet radiation. The sunscreen formulation may contain, forexample, an effective amount of PABA or zinc oxide to minimize skinexposure to ultraviolet radiation.

Combination therapies are also provided that combine the compounds offormula I and/or II (which includes salts thereof) with another agentthat treats vitiligo or another condition. Combination formulations forthe treatment of vitiligo may include combination formulations thatinclude a topical corticosteroid, such as a Group I, II, III, IV, V, VIor VII corticosteroid, for example any of the following:

Group I (very potent: up to 600 times stronger than hydrocortisone)

Clobetasol propionate 0.05% (Dermovate®)

Betamethasone dipropionate 0.25% (Diprolene®)

Halobetasol proprionate 0.05% (Ultravate®)

Diflorasone diacetate 0.05% (Psorcon®)

Group II

Fluocinonide 0.05% (Lidex®)

Halcinonide 0.05% (Halog®)

Amcinonide 0.05% (Cyclocort®)

Desoximetasone 0.25% (Topicort®)

Group III

Triamcinolone acetonide 0.5% (Kenalog®, Aristocort® cream)

Mometasone furoate 0.1% (Elocon® ointment)

Fluticasone propionate 0.005% (Cutivate®)

Betamethasone dipropionate 0.05% (Diprosone®)

Group IV

Fluocinolone acetonide 0.01-0.2% (Synalar®, Synemol®, Fluonid®)

Hydrocortisone valerate 0.2% (Westcort®)

Hydrocortisone butyrate 0.1% (Locoid®)

Flurandrenolide 0.05% (Cordran®)

Triamcinolone acetonide 0.1% (Kenalog®, Aristocort® A ointment)

Mometasone furoate 0.1% (Elocon® cream, lotion)

Group V

Triamcinolone acetonide 0.1% (Kenalog®, Aristocort® cream, lotion)

Fluticasone propionate 0.05% (Cutivate® cream)

Desonide 0.05% (Tridesilon®, DesOwen® ointment)

Fluocinolone acetonide 0.025% (Synalar®, Synemol® cream)

Hydrocortisone valerate 0.2% (Westcort® cream)

Group VI

Prednicarbate 0.05% (Aclovate® cream, ointment)

Triamcinolone acetonide 0.025% (Aristocort® A cream, Kenalog® lotion)

Fluocinolone acetonide 0.01% (Capex® shampoo, Dermasmooth®)

Desonide 0.05% (DesOwen® cream, lotion)

Group VII

Hydrocortisone 2.5% (Hytone® cream, lotion, ointment)

Hydrocortisone 1% (Many over-the-counter brands)

In some examples, the subject is diagnosed with a disorder in additionto vitiligo. For example, the subject presents with vitiligo and anotherpre-existing disorder, and is treated with the topical formulation thatcontains the compounds of formula I and/or II. The subject may betreated in combination or adjunctively with a therapeutic agent for thepre-existing disorder. If the pre-existing disorder is, for example, athyroid disorder, the subject may be treated with thyroid hormonereplacement.

In another example, a subject with vitiligo of the eye, or around theeye, may also be diagnosed with dry eyes and the combination therapy isadministered to the subject. In one example, the subject is found tohave a meibomitis that would be responsive to topical application ofcorticosteroids, such as a prednisolone acetate ophthalmic suspension1%. The compounds of formula I and/or II (which includes salts thereof)are suspended in the prednisolone formulation and instilled in orapplied to the eye 2 to 4 times a day. In other examples, if the dryeyes are associated with seasonal allergies or other inflammatoryconditions, the eye drops are administered with or in a formulation thatincludes antihistamines (such as pheniramine, emedastine, orazelastine), decongestants (such as tetrahydrozoline hydrochloride ornaphazoline), or a non-steroidal anti-inflammatory agent (such asnepafenac or ketorolac), corticosteroids (such as fluorometholone orloteprednol), mast cell stabilizers (such as azelastie, cromal,emedastine, ketotifen, Iodoxamine, nedocromil, olopatadine, orpemirolast). If the dry eyes are associated with an infectious bacterialcondition (such a meibomian gland infection or corneal infection) theeye drops are administered with or in a combination formulation cancontain appropriate antibiotics (such as ciprofloxacin, erythromycin,gentamicin, ofloxacin, sulfacetamine, tobramycin, or monofloxacin). Ifthe dry eyes are associated with a viral infection, the eye drops areadministered with or in a combination formulation with an anti-viralagent such as trifluridine or idoxuridine.

Example 9 Topical Applicators and Dosage Forms

The compositions of the invention may be used in an application devicethat permits application of the composition to a target site on the skinwithout applying the composition to non-target site areas of the skin.For example, a device may be employed that allows the composition to beapplied without first applying the composition to one's fingers.Suitable devices include spatulas, swabs, syringes without needles, andadhesive patches. Use of spatulas or swabs, or the like may require thedevice to be inserted into a container containing the composition. Usingsyringes or adhesive patches may be accomplished by filling the syringeor patch with the composition. The composition may then be topicallyspread by the spatulas or swabs, or may be expelled from the syringesonto the person's skin.

In one embodiment of the invention, the composition containing thecompound and the enhancing agent is provided in an adhesive patch. Someexamples of adhesive patches are well known. For example, see U.S. Pat.Nos. Des. 296,006; 6,010,715; 5,591,767; 5,008,110; 5,683,712;5,948,433; and 5,965,154. Such patches generally have an adhesive layer,which is applied to a person's skin, a depot or reservoir for holdingthe pharmaceutical agent, and an exterior surface that prevents leakageof the pharmaceutical from the depot. The exterior surface of a patch istypically non-adhesive.

In accordance with the present invention, the compound for treatingvitiligo is incorporated into the patch so that the compound remainsstable for extended periods of time. The compound may be incorporatedinto a polymeric matrix that stabilizes it, and permits the compound todiffuse from the matrix and the patch. The compound may also beincorporated into the adhesive layer of the patch so that once the patchis applied to the skin the compound may diffuse on to the skin or eveninto or through the skin. In accordance with such an embodiment, theadhesive preferably comprises an enhancing agent, as disclosed herein.In one embodiment, the adhesive layer may be heat activated wherebytemperatures of about 37° C. cause the adhesive to slowly liquefy sothat the compound diffuses out of the patch and on to, into, or throughthe skin. The adhesive may remain tacky when stored at less than 37° C.,and once applied to the skin, the adhesive loses its tackiness as itliquefies. The administration of the compound is complete once the patchno longer adheres to the skin.

Alternatively, the compound may be provided in one or more wells orpockets disposed near the surface of the patch that will contact theskin. In one embodiment, the compound is stored in the wells in a dried,or lyophilized state. Storing such patches in a cooled atmosphere (e.g.,about 4° C.) maintains the stability of the compound. A patch may beremoved from the cool atmosphere when needed, and applied to a person'sskin where the compound may be solubilized upon mixing with fluid, suchas water or saline. The fluid may be provided separately or as acomponent of the patch. For example, fluid may be provided on a person'sskin so that when the patch containing the dried compound interacts withthe fluid, the compound is exposed to the fluid and is solubilized. Thesolubilized compound may then be able to be absorbed by the skin. Asanother example, the patch may contain one or more wells or pockets tohold fluid in the patch. The fluid may be forced from the wells orpockets to cause the fluid to mix with the dried compound. For example,the fluid may be provided in a pocket in the patch, and in someembodiments contains an agent for enhancing or activating the compound.Pressure exerted on the patch causes the pocket to rupture and releasethe fluid so that it mixes with the dried compound. The compositioncontaining the compound may thus diffuse out of the patch. In anotherexample, a fluid such as a gel or creams that contains water may beapplied to the skin at a target site. The patch containing the driedcompound is then applied to the skin where the fluid mixes with thecompound and the composition moves out of the patch and on to the skin.

In patches containing wells of dried compound, the wells are sealed sothat the compound remains in the wells until the compound isadministered. Accordingly, the wells are sealed with a membrane or filmthat prevents the compound from diffusing from the wells in thecompound's dry state, but that permits the compound to diffuse from thewells when it is solubilized. The membrane may either be porous ornonporous. In one embodiment, the membrane comprises cellulose orstarch, and more particularly, the membrane may contain polyvinylalcohol, polyethylene oxide, and hydroxypropyl methyl cellulose. Themembrane is thin (ranging in thickness from about 1 μm to about 1 mm)and dissolves upon contacting liquid. Thus, gel or cream placed on theperson's skin or fluid directed from a pocket in the patch may contactthe cellulose membrane and cause the membrane to dissolve. Afterdissolving, the fluid mixes with the dried compound and solubilizes thecompound. The composition then diffuses out of the patch and on to thesubject's skin.

Additionally, the transdermal patch may include a plurality of smallneedles that extend through the stratum corneum, but do not extend intothe dermis to rupture blood vessels. The needles may be between 20 μmand 1 mm long when extending from the dermal surface of the patch. Thus,the needles extend through the stratum corneum, but terminate before thedermis where the capillary beds are located. The needles may be solid orhollow. Hollow needles may have a lumen extending along their length sothat the composition can pass from the depot in the patch to the end ofthe needle in the epidermis. Solid needles may be used to permit thecomposition to diffuse along the outer surface of the needle into theepidermis.

In use, the topical applicator is adhesively applied to a target area ofthe skin that has one or more depigmented patches, and the applicator isleft in place until the compound in the patch is administered. Thetopical applicator (such as a patch) provides sustained release of thedrug over a prolonged period of time, such as several hours, or even atleast a day or longer.

Example 10 Other Dosage Forms and Additives

The topical formulation may be prepared in a variety of forms. Solidsare generally firm and non-pourable and commonly are formulated as a baror stick, or in particulate form; solids may be opaque or transparent,and optionally may contain solvents (including water and alcohol),emulsifiers, moisturizers, emollients, fragrances, dyes/colorants,preservatives and active ingredients. Creams and lotions are oftensimilar to one another, differing mainly in their viscosity (creams aretypically thicker and more viscous than lotions); both lotions andcreams may be opaque, translucent or clear and often containemulsifiers, solvents (including water and alcohol) and viscosityadjusting agents. Lotions and creams also may optionally containmoisturizers and emollients (especially in the case of skin careproducts), as well as fragrances, dyes/colorants, preservatives andactive ingredients. Gels/serums may be prepared with a range ofviscosities, from thick (high viscosity) to thin (low viscosity) anddiffer principally from lotions and creams in that gels/serums areusually clear rather than opaque. Like lotions and creams, gels/serumsoften contain emulsifiers, solvents (including water and alcohol) andviscosity adjusters, and may also contain moisturizers and emollients,fragrances, dyes/colorants, preservatives and active ingredients.Aqueous liquids are thinner than creams, lotions or gels, and aregenerally transparent; liquids usually do not contain emulsifiers.Liquid topical products often contain other solvents in addition towater (including alcohol) and may also contain viscosity adjusters,moisturizers and emollients, fragrances, dyes/colorants/pigments,preservatives and active ingredients.

Suitable emulsifiers for use in the formulations include, but are notlimited to, Incroquat™ Behenyl TMS (behentrimonium methosulfate,cetearyl alcohol), non-ionic emulsifiers like polyoxyethylene oleylether, PEG-40 stearate, ceteareth-12 (e.g., Eumulgin® B-1 manufacturedby Henkel), ceteareth-20 (e.g., Eumulgin® B-2 manufactured by Henkel),ceteareth-30, Lanette O (manufactured by Henkel; ceteareth alcohol),glyceryl stearate (e.g., Cutina® GMS manufactured by Henkel), PEG-100stearate, Arlacel™ 165 (glyceryl stearate and PEG-100 stearate),steareth-2 and steareth-20, or combinations/mixtures thereof, as well ascationic emulsifiers like stearamidopropyl dimethylamine andbehentrimonium methosulfate, or combinations/mixtures thereof. Inaddition, cationic emulsifiers are preferably combined or mixed withnon-ionic emulsifiers in order to form stable emulsion product formscontaining high strontium salt concentrations.

Suitable secondary active ingredients for use in the formulationsinclude, but are not limited to, alpha hydroxy acids, sunscreens,antiperspirants, anti-acne drugs, vitamins (especially vitamins A and C)and minerals, and various prescription and over-the-counter medications.The compositions disclosed herein can have multiple active ingredientswithin the same topical formulation, and combinations of activeingredients such as those listed above may be used, as appropriate forthe condition or conditions being treated.

Suitable fragrances and colors, such as FD&C Red No. 40 and FD&C YellowNo. 5, may be used in the formulations of the present invention. Otherexamples of fragrances and colors suitable for use in topical productsare known in the art.

Other suitable additional and adjunct ingredients which may be includedin the formulations include, but are not limited to, abrasives,absorbents, anti-caking agents, anti-foaming agents, anti-static agents,astringents (e.g., witch hazel, alcohol, and herbal extracts such aschamomile extract), binders/excipients, buffering agents, chelatingagents (e.g., Versene EDTA), film forming agents, conditioning agents,opacifying agents, pH adjusters (e.g., citric acid and sodiumhydroxide), and protectants. Examples of each of these ingredients, aswell as examples of other suitable ingredients in topical productformulations, may be found in publications by The Cosmetic, Toiletry,and Fragrance Association (CTFA). See, e.g., CTFA Cosmetic IngredientHandbook, 2nd edition, eds. John A. Wenninger and G. N. McEwen, Jr.(CTFA, 1992).

Also, a variety of product types, including cosmetics, may be formulatedin each of the forms described above (i.e., solids, creams, lotions,gels, and liquids). For example, cleansers (for face and body),shampoos/conditioners, hair treatments/dyes/perms/straighteners,antiperspirants/deodorants, make-up products, and other facial, hand andbody products may be formulated in any of the five major product forms:solids, creams, lotions, gels, or liquids. Common solid form productsinclude cosmetics such as lipsticks, blushes and rouges, makeupproducts, antiperspirant and deodorant sticks, and cleansers such as barsoap and powder detergents. Other examples of solid form productsinclude lozenges and suppositories for the treatment of cutaneous lupuslesions of the mucous membranes (such as the mouth or anus). Commoncream and lotion form products include alpha-hydroxy acid (AHA)products, moisturizing products and sunscreens, shampoos/conditionersand other hair care products, and cosmetics like concealers andfoundations. Common gel products include shaving gels and aftershaves.Common liquid form products include anti-acne solutions, aftershaves,gargles/mouthwashes, and toners/bracers/skin conditioners.

Other methodologies and materials for preparing formulations in avariety of forms are also described in Anthony L. L. Hunting (ed.), “AFormulary of Cosmetic Preparations (Vol. 2)—Creams, Lotions and Milks,”Micelle Press (England, N.J. 1993). See, for example, Chapter 7, pp.5-14 (oils and gels); Chapter 8, pp. 15-98 (bases and emulsions);Chapter 9, pp. 101-120 (“all-purpose products”); Chapter 10, pp. 121-184(cleansing masks, creams, lotions); Chapter 11, pp. 185-208 (foundation,vanishing and day creams); Chapter 12, pp. 209-254 (emollients); Chapter13, pp. 297-324 (facial treatment products); Chapter 14, pp. 325-380(hand products); Chapter 15, pp. 381-460 (body and skin creams andlotions); and Chapter 16, pp. 461-484 (baby products); the contents ofwhich are incorporated herein by reference.

Example 11 An Exemplary Topical Formulation

The topical formulation may be prepared in a variety of strengths andusing a variety of excipient concentrations as described herein. Table 3is a list of the excipients used in this example, and without beinglimited to any particular theory, the function of each excipient.

TABLE 3 Excipient Function PEG400, Glycofurol Solvent PEG8000, PEG4500,PEG3350 Topical Base Tefose ® 63, Span ®, Myrj ®, TPGS Surfactant DMI,PG Penetration enhancer H₂O Emollient BHT Antioxidant Caramel ColorAdditive

With reference to Table 3, PEG400 employed in working examples was SuperRefined Polyethylene Glycol 400, commercially available from Croda Inc.,Edison N.J. Likewise, Super Refined dimethyl isosorbide (DMI), alsoavailable from Croda Inc. typically was used in these examples.

To prepare the formulations, excipients and compound I was added to aglass container, and heated and/or sonicated at 65° C. to 70° C. todissolve API completely. The sample is then cooled to room temperature.The ingredients for two exemplary formulations prepared by this methodare set forth below in Tables 4 and 5.

TABLE 4 Weight (g) Component Grade Weight % per kg Compound I GMP 3.0 30Super Refined Polyethylene Glycol 400 NF 39.95 399.5 Polyethylene Glycol4500 NF 32.0 320 Butylated Hydroxytoluene, Granular NF 1.0 10 MYRJS100-PA-SG — 5.0 50 Super Refined Dimethyl Isosorbide — 15.0 150Purified Water USP 4.0 40 Caramel NF 0.05 0.5 Total 100 1000

TABLE 5 Weight (g) Component Grade Weight % per kg Compound I GMP 6.0 60Super Refined Polyethylene Glycol 400 NF 33.95 339.5 Polyethylene Glycol4500 NF 35.0 350 Butylated Hydroxytoluene, Granular NF 1.0 10 MYRJS100-PA-SG — 5.0 50 Super Refined Dimethyl Isosorbide — 15.0 150Purified Water USP 4.0 40 Caramel NF 0.05 0.5 Total 100 1000

I claim:
 1. A method of treating vitiligo, comprising administering to asubject a therapeutically effective amount of a compound of formula I,formula II, or both


2. The method of claim 1, wherein the compound is in the form of aprodrug, a hydrate, a solvate, an N-oxide, or a pharmaceuticallyacceptable salt.
 3. The method of claim 1, wherein the subject isidentified as having vitiligo.
 4. The method of claim 1, wherein thesubject is identified as being at risk of developing vitiligo.
 5. Themethod of claim 1, wherein the therapeutically effective amount rangesfrom about 0.0001 mg/kg/day to about 100 mg/kg/day.
 6. The method ofclaim 1, wherein the compound is administered as a topical formulationcomprising from 0.0001 to 10% (w/w) of the compound of formula I and/orII.
 7. The method of claim 1, wherein the subject has one or moredepigmented skin areas, and the compound is applied topically to atleast one of the depigmented skin areas.
 8. The method of claim 7,wherein the compound is also administered systemically to the subject.9. The method of claim 1, wherein the compound is administeredsystemically to the subject.
 10. The method of claim 1, wherein thesubject has vitiligo of the eye, and the compound is administeredocularly to the patient to treat the vitiligo.
 11. The method of claim1, wherein the compound is a salt of compound II.
 12. The method ofclaim 11, wherein the salt of compound II is selected from a sodiumsalt, a potassium salt, a calcium salt, an arginine salt, choline salt,and combinations thereof.
 13. The method of claim 1, wherein thecompound is administered either in combination or adjunctively with asecond therapeutic.
 14. The method of claim 13, wherein the secondtherapeutic is an anti-inflammatory, an antihistamine, an antibiotic, anantiviral medication, systemic phototherapy, psoralen photochemotherapy,excimer laser therapy, thyroid hormone replacement medication, or anycombination thereof.
 15. A pharmaceutical formulation comprising atherapeutically effective amount of a compound of formula I, formula II,or both

wherein the formulation is a topical dosage form formulated foradministration to a subject identified as having vitiligo or as being atrisk of developing vitiligo.
 16. The pharmaceutical formulation of claim15, wherein the compound is in the form of a prodrug, a hydrate, asolvate, an N-oxide, or a pharmaceutically acceptable salt.
 17. Thepharmaceutical formulation of claim 15, wherein the topical dosage formcomprises a solution, a gel, an ointment, a cream or a suspension. 18.The pharmaceutical formulation of claim 15, wherein the compound ispresent in a concentration ranging from 0.0001 to 10% (w/w).
 19. Thepharmaceutical formulation of claim 15, wherein the topical dosage formcomprises an adhesive applicator.
 20. A kit comprising a pharmaceuticalformulation comprising a therapeutically effective amount of a compoundof formula I, formula II, or both

or a pharmaceutically acceptable salt form thereof, formulated foradministration to the skin of a subject identified as having vitiligo oras being at risk of developing vitiligo.